Nucleoside and nucleotide analogues bearing a quaternary all-carbon stereogenic center at the 2&#39; position and methods of use as a cardioprotective agent

ABSTRACT

Nucleoside and nucleotide analogues that can be used as cardioprotective agents are provided. The nucleosides and nucleotide analogues comprise tetrahydrofuranyl or tetrahydrothienyl moieties with quaternary stereogenic all-carbon centers at the 2′ position and a phosphonate ester at the 5′ position.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 National Phase Entry Applicationfrom PCT/CA2017/051095, filed Sep. 18, 2017, which claims benefit, under35 U.S.C. § 119(e), of U.S. provisional application Ser. No. 62/395,401filed on Sep. 16, 2016, U.S. provisional application Ser. No. 62/395,411filed on Sep. 16, 2016, and U.S. provisional application Ser. No.62/395,430 filed on Sep. 16, 2016. All documents above are incorporatedherein in their entirety by reference.

FIELD OF THE INVENTION

The present invention relates to nucleoside and nucleotide analogues.

BACKGROUND OF THE INVENTION

Nucleosides and nucleotides are one of the most important classes ofmolecules in biology. Nucleotides are the monomeric units of RNA andDNA. They are also required for other numerous functions in the cell.For example, they are involved in phosphate transfer reactions (ATP) ascoenzymes (for example NAD^(T), FAD and coenzyme A) and activatedintermediates (S-adenosylmethionine). The nucleosides are transported byknown equilibrative nucleoside transporters (hENTs) and humanconcentrative nucleoside transporters (hCNTs) in the cells.

Various nucleotide analogues are used as pharmaceutical agents. Forexample, some nucleoside analogues are utilized as antitumor agents,interfering with the synthesis of DNA or RNA in dividing cancerouscells. Various nucleotide analogues in their phosphorylated forms arealso included in antisense RNA, siRNA or micro RNA to control thetranscription and translation of genes. Various nucleoside or nucleotideanalogues also interfere with various purinergic receptors (P1, P2Y andP2X) as either an agonist or an antagonist.1,2 Nucleoside analogues suchas nitrobenzylmercaptopurine (NBMPR) can inhibit nucleoside transporters(e.g. ENT-1 and ENT-2).

On another subject, heart failure (HF) occurs when the heart is unableto pump sufficiently to maintain blood flow to meet the body's needs. Inheart failure, the heart ventricles may become stiff and do not fillproperly between beats. In some cases of heart failure, the heart musclemay become damaged and weakened, and the ventricles dilate to the pointthat the heart can't pump blood efficiently throughout the body. Overtime, the heart can no longer keep up with the normal demands placed onit to pump blood to the body. The term “congestive heart failure” comesfrom blood backing up into—or congesting—the liver, abdomen, lowerextremities and lungs. However, not all heart failure is congestive.

Heart failure has many causes and underlying risk factors. The mostcommon is damage to the heart caused by a myocardial infarction. Asignificant other common cause is untreated high blood pressure for along period. In fact, HF often develops after the heart has been damagedor weakened by other conditions such as:

-   -   Coronary artery disease and heart attack. Coronary artery        disease is the most common form of heart disease and the most        common cause of heart failure. Over time, arteries that supply        blood to the heart muscle narrow from atherosclerosis, which can        cause reduced blood flow to the heart. A heart attack occurs if        the plaques formed by the fatty deposits in the arteries rupture        and form a blood clot, which may block blood flow to an area of        the heart muscle, weakening the heart's pumping ability and        often leaving permanent damage. If the damage is significant, it        can lead to a weakened heart muscle.    -   High blood pressure (hypertension). When blood pressure is high,        the heart has to work harder than it should to circulate blood        throughout the body. Over time, the heart muscle may become        thicker to compensate. Eventually, the heart muscle may become        either too stiff or too weak to effectively pump blood.    -   Faulty heart valves. A damaged valve—due to a heart defect,        coronary artery disease or heart infection—forces the heart to        work harder to keep blood flowing as it should. Over time, this        extra work can weaken the heart.    -   Cardiomyopathy. Heart muscle damage (cardiomyopathy) can have        many causes, including several diseases, infections, alcohol        abuse and the toxic effect of drugs, such as cocaine or some        drugs used for chemotherapy. Genetic factors play an important        role in several types of cardiomyopathy, such as dilated        cardiomyopathy, hypertrophic cardiomyopathy, arrhythmogenic        right ventricular cardiomyopathy, left ventricular noncompaction        and restrictive cardiomyopathy.    -   Myocarditis. Myocarditis is an inflammation of the heart muscle.        It is most commonly caused by a virus and can lead to left-sided        heart failure.    -   Congenital heart defects. If the heart and its chambers or        valves haven't formed correctly, the healthy parts of the heart        have to work harder to pump blood through the heart, which, in        turn, may lead to heart failure.    -   Heart arrhythmias. Abnormal heart rhythms may cause the heart to        beat too fast, which creates extra work for the heart. Over        time, the heart may weaken, leading to heart failure. A slow        heartbeat may prevent the heart from getting enough blood out to        the body and may also lead to heart failure.    -   Other diseases. Chronic diseases—such as diabetes, HIV,        hyperthyroidism, hypothyroidism, or a buildup of iron        (hemochromatosis) or protein (amyloidosis)—also may contribute        to heart failure. Causes of acute heart failure include viruses        that attack the heart muscle, severe infections, allergic        reactions, blood clots in the lungs, the use of certain        medications or any illness that affects the whole body.

The “ejection fraction” is an important measurement of how well a heartis pumping and is used to help classify heart failure and guidetreatment. In a healthy heart, the ejection fraction is 50 percent orhigher—meaning that more than half of the blood that fills the ventricleis pumped out with each beat. But heart failure can occur even with anormal ejection fraction. This happens if the heart muscle becomes stifffrom conditions such as high blood pressure.

At present, there are no effective therapies for the prevention ortreatment of HF and about 50% of patients with HF die within 5 years.

On yet another subject, progress in cancer therapeutics over the pastdecades has been remarkable in improving survival rates and prolongingpatients' life. However, it has also revealed undesirable consequencessuch as the significant increase in cardiovascular disease. In survivorsof childhood and adolescent cancers, cancer treatment-inducedcardiotoxicity is the third leading cause of mortality, behindrecurrence and other malignancies. In adults, heart problems are themost reported post-cancer treatment issues in female survivors afterarthritis-osteoporosis while in males it is the number one problem in5-10 year survivors.^(3,4) Chemotherapy associated cardiac toxicityranges from asymptomatic subclinical changes to life-threatening eventslike congestive heart failure.

Anthracyclines are known to induce irreversible cardiac damage and theircardiotoxicity is further enhanced by other agents. Moreover, newer,more targeted therapies, including receptor specific monoclonalantibodies and tyrosine kinase inhibitors (TKIs) are starting to beassociated with cardiac dysfunction in cancer survivors. In the case ofbreast cancer, the incidence and severity of the anthracyclineDoxorubicin (DOX) cardiotoxicity are known to be dose-dependent,increasing with cumulative doses and the presence of other drugs. Theincidence of congestive heart failure goes up to 7% at 550 mg/m² and 20%of cumulative doses over 700 mg/m². DOX cardiotoxicity is even morewidespread in patients receiving high doses of cyclophosphamide,paclitaxel or Trastuzumab (TRZ), a monoclonal antibody against theextracellular domain of the human epidermal growth factor receptor 2protein (HER2). The latter is used in HER2 positive breast cancercombined with DOX. Unfortunately, it is now recognized that TRZpotentiates the cardiotoxic side effects of DOX. Recent studiesindicated that nearly 1 in 4 women will develop a drug-inducedcardiotoxicity. Biomarkers such as troponin-I and N-terminal pro-brainnatriuretic peptide (NT-proBNP) combined to tissue Doppler imaging (TDI)are becoming recognized as early markers for subclinical latecardiotoxicity.⁵ Irreversible DOX cardiotoxicity is due to its inductionof cardiomyocyte death. Cardiomyocytes, the contractile cells of theheart have limited regenerative potential and their loss leads to heartfailure.

Drug-induced cardiotoxicity can lead to heart failure which ischaracterized by cardiac remodeling and decreased ejection fraction(EF). These abnormalities contribute to inadequate cardiac output, poororgan perfusion, activation of the renin angiotensin-aldosterone system(RAAS) and the sympathetic nervous system (SNS).

Unlike other cell types, postnatal cardiomyocytes—which represent lessthan 30% of the cell number, but nearly 85% of the heart mass—becometerminally differentiated and essentially lose their ability to undergoproliferative growth. Loss of cardiomyocytes in the contractile unit ofthe heart leads to irreversible cardiac remodeling and dysfunction (seeFIG. 1). Cardiomyocyte loss is a major feature of human HF and was shownto be sufficient to trigger HF in a variety of experimental animalmodels.⁶⁻⁸ The limited regenerative ability of postnatal cardiomyocytesmeans that their response to stressors generally involves hypertrophy ordeath.⁹

Despite its vital importance, the mechanisms that control cardiomyocytesurvival remain poorly understood. Maintaining energy metabolism andmitochondrial function is critical. Upregulation of oxidative stressgenes negatively affects the mitochondria, and leads to cardiacdysfunction. Conversely, anti-apoptotic BCL2 or the mitochondrialbiogenesis PGC-1 co-activator proteins promote mitochondrial functionand are essential for cardiomyocyte survival in response to stressors.Thus, several genetic programs controlling energy metabolism,contractility, and stress response, need to be coordinately regulated tomaintain cardiomyocyte survival and cardiac homeostasis. GATA4 is amaster regulator of the genetic program required for cardiomyocytesurvival and adaptive stress response.^(6,10) Mice with 50% reduction inGATA4 are hypersensitive to DOX cardiotoxicity. In culturedcardiomyocytes, DOX treatment leads to rapid depletion of GATA4 andgenetic upregulation of GATA4 prevents DOX cardiotoxicity.Interestingly, Imatinib (a TKI) was also found to induce cardiomyocyteapoptosis and mitochondrial dysfunction through a GATA4-dependentpathway. The mechanism by which GATA4 prevents DOX-induced apoptosis isnot fully understood. On the other hand, caspase 1—which is induced atearly stages of mitochondrial stress—directly targets GATA4 fordegradation.¹¹ Thus, a reinforcing feedback loop may exist between GATA4and energy metabolism to maintain cardiomyocyte cell survival. GATA4activates numerous pro-survival genes including BCL2 familymembers.^(7,11)

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided:

-   -   1. A compound of formula:

-   -   -   or a pharmaceutically acceptable salt thereof,        -   wherein:            -   A and B are C₁-C₆ alkyl, mono- to per-halo C₁-C₆ alkyl,                —(CH₂)_(n)M, —C≡N, or

-   -   -   -    with the proviso that:                -   A is different from B,                -   when one of A and B is methyl, the other is not                    —CF₃, and                -   when one of A and B is C₂-C₆ alkyl, the other is not                    C₂-C₆ fluoroalkyl;            -   n is 1 to 3;            -   R₁ is

-   -   -   -   R₂ is the same or different, preferably the same, and is                C₁-C₆ alkyl;            -   M is —OR₃, —SR₃, aryl, —C(O)OR₃, or —OC(O)R₄;            -   R₃ is —H, C₁-C₆ alkyl, aryl, aryl-C₁-C₆ alkyl, C₁-C₆                alkylaryl, wherein each of the alkyl and aryl groups is                optionally substituted with one or more groups selected                from halo, mono- to per-halo C₁-C₆ alkyl, —CN, —C(O)OH,                —C(O)OR₄, —N₃, —C₁-C₆ alkyl-C(O)OR₄, —CF₃, —C₁-C₆                alkyl-N₃, and —SiF₅;            -   R₄ is C₁-C₆ alkyl, aryl, heteroaryl, C₁-C₆ alkylaryl,                aryl-C₁-C₆ alkyl, wherein each of the alkyl, aryl and                heteroaryl groups is optionally substituted with one or                more groups selected from halo, —CN, alkynyl,                alkynyloxy, —C(O)OH, —N₃, —CF₃, —C₁-C₆ alkyl-N₃, —SiF₅,                —NH₂, and —NHR₃;            -   C and D are independently —H, halo, azido, —OR₃, —CN, or                —CF₃;            -   X is O or S; and            -   Base is:

-   -   -   -   R₅ is —H, —C(O)—C₁-C₄ alkyl, aryl, alkylaryl, or                arylalkyl, wherein each of the alkyl and aryl group is                optionally substituted with one or more groups selected                from halo, —R₄, —OF₃, and —N₃.

    -   2. The compound of item 1 being of the formulae:

-   -   3. The compound of item 2 being of the formulae:

-   -   4. The compound of item 3 being of the formulae:

-   -   5. The compound of any one of items 1 to 4, wherein A and B are        C₁-C₆ alkyl, —(CH₂)_(n)M, —C≡N, or

preferably C₁-C₆ alkyl or —(CH₂)_(n)M.

-   -   6. The compound of any one of items 1 to 5, wherein one of A or        B is C₁-C₆ alkyl while the other is —(CH₂)_(n)M, —C≡N, or

preferably —(CH₂)_(n)M.

-   -   7. The compound of any one of items 1 to 6, wherein A is C₁-C₆        alkyl and B is —(CH₂)_(n)M, —C≡N, or

preferably —(CH₂)_(n)M.

-   -   8. The compound of any one of items 1 to 7, wherein, in A and B,        the C₁-C₆ alkyl is methyl.    -   9. The compound of any one of items 1 to 8, wherein n is 1.    -   10. The compound of any one of items 1 to 9, wherein M is —OR₃        or —OC(O)R₄.    -   11. The compound of any one of items 1 to 10, wherein M is        —OC(O)R₄.    -   12. The compound of any one of items 1 to 10, wherein M is —OR₃.    -   13. The compound of any one of items 1 to 12, wherein R₃ is —H,        C₁-C₆ alkyl, or aryl-C₁-C₆ alkyl, wherein preferably the aryl of        the aryl-C₁-C₆ alkyl is optionally substituted with one or more:        -   halo,        -   mono- to per-halo C₁-C₆ alkyl,        -   —N₃, and/or        -   —C₁-C₆ alkyl-N₃.    -   14. The compound of any one of items 1 to 13, wherein the halo        optionally substituting the aryl of the aryl-C₁-C₆ alkyl in R₃        is —F.    -   15. The compound of any one of items 1 to 14, wherein the mono-        to per-halo C₁-C₆ alkyl optionally substituting the aryl of the        aryl-C₁-C₆ alkyl in R₃ is per-halo C₁-C₆ alkyl.    -   16. The compound of any one of items 1 to 15, wherein the mono-        to per-halo C₁-C₆ alkyl optionally substituting the aryl of the        aryl-C₁-C₆ alkyl in R₃ is mono- to per-halo methyl, preferably        —CF₃.    -   17. The compound of any one of items 1 to 16, wherein the —C₁-C₆        alkyl-N₃ optionally substituting the aryl of the aryl-C₁-C₆        alkyl in R₃ is —CH₂—N₃.    -   18. The compound of any one of items 1 to 17, wherein the        aryl-C₁-C₆ alkyl in R₃ is benzyl optionally substituted with one        or more:        -   halo,        -   mono- to per-halo C₁-C₆ alkyl,        -   —N₃, and/or        -   —C₁-C₆ alkyl-N₃.    -   19. The compound of any one of items 1 to 18, wherein R₃ is —H,        methyl, isopropyl, benzyl,

-   -   20. The compound of any one of items 1 to 19, wherein R₃ is —H        or benzyl.    -   21. The compound of any one of items 1 to 20, wherein R₃ is —H.    -   22. The compound of any one of items 1 to 21, wherein, in        —OC(O)R₄ in M, R₄ is aryl or heteroaryl, the aryl and heteroaryl        being optionally substituted with one or more groups selected        from halo, —CN, alkynyl, alkynyloxy, —C(O)OH, —N₃, —CF₃, —C₁-C₆        alkyl-N₃, —SiF₅, —NH₂, and —NHR₃.    -   23. The compound of any one of items 1 to 22, wherein, in        —OC(O)R₄ in M, the aryl in R₄ is benzoylphenyl.    -   24. The compound of any one of items 1 to 23, wherein, in        —OC(O)R₄ in M, the heteroaryl in R₄ is indole-5-carbonylphenyl.    -   25. The compound of any one of items 1 to 24, wherein, in        —OC(O)R₄ in M, the aryl or heteroaryl in R₄ is substituted with        alkynyl or alkynyloxy.    -   26. The compound of any one of items 1 to 25, wherein, in        —OC(O)R₄ in M, the aryl in R₄ is substituted with alkynyloxy.    -   27. The compound of any one of items 1 to 26, wherein, in        —OC(O)R₄ in M, the heteroaryl in R₄ is substituted with alkynyl.    -   28. The compound of any one of items 1 to 27, wherein, in        —OC(O)R₄ in M, the alkynyloxy optionally substituting the aryl        or heteroaryl in R₄ is prop-2-yn-1-yloxy (—O—CH₂—C≡CH).    -   29. The compound of any one of items 1 to 28, wherein, in        —OC(O)R₄ in M, the alkynyl optionally substituting the aryl or        heteroaryl in R₄ is prop-2-yn-1-yl (—CH₂—C≡CH).    -   30. The compound of any one of items 1 to 29, wherein, in        —OC(O)R₄ in M, R₄ is

-   -   31. The compound of any one of items 1 to 30, wherein C and D        are independently —H, halo, or —OR₃.    -   32. The compound of any one of items 1 to 31, wherein C and D        are independently —H, halo, or —OH.    -   33. The compound of any one of items 1 to 32, wherein one of C        or D is —H and the other is halo or —OR₃, wherein —OR₃        preferably represents —OH.    -   34. The compound of any one of items 1 to 33, wherein C is —H        and D is halo or —OR₃, wherein —OR₃ preferably represents —OH.    -   35. The compound of any one of items 1 to 34, wherein one of C        or D is —H and the other is —OR₃, wherein —OR₃ preferably        represents —OH.    -   36. The compound of any one of items 1 to 35, wherein C is —H        and D is —OR₃, wherein —OR₃ preferably represents —OH.    -   37. The compound of any one of items 1 to 36, wherein the halo        in C and D is —F.    -   38. The compound of any one of items 1 to 37, wherein X is O.    -   39. The compound of any one of items 1 to 38, wherein R₅        represents —H, —C(O)—C₁-C₄ alkyl, arylalkyl, or aryl, wherein        the aryl group is optionally substituted with one or more groups        selected from halo, —R₄, —CF₃, and —N₃.    -   40. The compound of any one of items 1 to 39, wherein R₅        represents —H, —C(O)—C₁-C₄ alkyl, arylalkyl, or aryl, wherein        each of the aryl groups is optionally substituted with one or        more groups selected from halo and —R₄.    -   41. The compound of any one of items 1 to 40, wherein the alkyl        group in —C(O)—C₁-C₄ alkyl in R₅ is propyl.    -   42. The compound of any one of items 1 to 41, wherein the aryl        group of the arylalkyl in R₅ is optionally substituted with one        or more —R₄.    -   43. The compound of any one of items 1 to 42, wherein the aryl        group of the arylalkyl in R₅ is unsubstituted.    -   44. The compound of any one of items 1 to 43, wherein the        arylalkyl in R₅ is benzyl.    -   45. The compound of any one of items 1 to 44, wherein the aryl        in R₅ is optionally substituted with one or more, preferably        one, F or —CF₃, preferably —CF₃.    -   46. The compound of any one of items 1 to 45, wherein the aryl        in R₅ is phenyl.    -   47. The compound of any one of items 1 to 46, wherein R₅        represents —H, —C(O)-propyl, benzyl, or p-trifluoromethylphenyl.    -   48. The compound of any one of items 1 to 47, wherein base is:

-   -   49. The compound of any one of items 1 to 48, wherein base is:

-   -   50. The compound of any one of items 1 to 50, wherein base is:

-   -   51. The compound of any one of items 1 to 48, wherein base is:

-   -   52. The compound of any one of items 1 to 48, wherein base is

-   -   53. The compound of any one of items 1 to 48, wherein base is

-   -   54. The compound of any one of items 1 to 48, wherein base is

-   -   55. The compound of any one of items 1 to 48, wherein base is

-   -   56. The compound of any one of items 1 to 48, wherein base is

-   -   57. The compound of any one of items 1 to 48, wherein base is

-   -   58. The compound of any one of items 1 to 48, wherein base is

-   -   59. The compound of any one of items 1 to 48, wherein base is

-   -   60. The compound of item 59, wherein R₅ is arylalkyl, wherein        the aryl group is optionally substituted with one or more groups        selected from halo, —R₄, —CF₃, and —N₃.    -   61. The compound of item 60, wherein R₅ is arylalkyl, wherein        the aryl group is optionally substituted with —R₄.    -   62. The compound of item 61, wherein R₅ is benzyl optionally        substituted with —R₄.    -   63. The compound of item 62, wherein R₅ is unsubstituted benzyl.    -   64. The compound of any one of items 1 to 48, wherein base is

-   -   65. The compound of any one of items 1 to 48, wherein base is

-   -   66. The compound of item 65, wherein R₅ represents —C(O)—C₁-C₄        alkyl or aryl optionally substituted with one or more groups        selected from halo, —R₄, —CF₃, and —N₃.    -   67. The compound of item 66, wherein R₅ represents aryl        optionally substituted with one or more groups selected from        halo, —R₄, —CF₃, and —N₃.    -   68. The compound of item 67, wherein R₅ represents aryl        optionally substituted with —CF₃.    -   69. The compound of item 68, wherein R₅ represents phenyl        optionally substituted with —CF₃.    -   70. The compound of item 69, wherein R₅ represents        p-trifluoromethylphenyl.    -   71. The compound of item 66, wherein R₅ represents —C(O)—C₁-C₄        alkyl.    -   72. The compound of item 71, wherein R₅ represents —C(O)-propyl.    -   73. The compound of item 72, wherein base is

-   -   74. The compound of any one of items 1 to 73, wherein R₁ is

-   -   75. The compound of any one of items 1 to 73, wherein R₁ is

-   -   76. The compound of any one of items 1 to 75, wherein R₂ is        methyl, ethyl, isopropyl or tert-butyl.    -   77. The compound of any one of items 1 to 76, wherein R₂ is        ethyl or isopropyl.    -   78. The compound of any one of items 1 to 77, wherein R₂ is        ethyl.    -   79. The compound of item 1 being:

-   -   -   or a pharmaceutically acceptable salt thereof to the            subject.

    -   80. The compound of item 79 being:

-   -   -   or a pharmaceutically acceptable salt thereof.

    -   81. The compound of item 80 being:

-   -   -   or a pharmaceutically acceptable salt thereof.

    -   82. The compound of item 81 being

or a pharmaceutically acceptable salt thereof.

-   -   83. A pharmaceutical composition comprising a pharmaceutically        acceptable carrier, excipient or diluent and the compound of any        one of items 1 to 82 or a pharmaceutically acceptable salt        thereof.    -   84. A method of providing cardioprotection in a subject in need        thereof, the method comprising administering the compound of any        one of items 1 to 82 or a pharmaceutically acceptable salt        thereof to the subject.    -   85. A method of preserving, reducing deterioration of, and/or        improving a cardiac function of a heart that has been subjected,        is subjected, or will be subjected to a cardiac insult, the        method comprising administering the compound of any one of items        1 to 82 or a pharmaceutically acceptable salt thereof to a        subject in need thereof.    -   86. The method of item 84, wherein said cardiac function is        ejection fraction or cardiac contractility.    -   87. A method of preventing, reducing, and/or reversing heart        damage due to a cardiac insult, the method comprising        administering the compound of any one of items 1 to 82 or a        pharmaceutically acceptable salt thereof to a subject in need        thereof.    -   88. The method of item 87, wherein the heart damage includes        abnormal cardiomyocyte apoptosis, cardiac remodeling including        changes in heart wall thickness, decrease in ejection fraction,        poor organ perfusion, and/or loss of cardiac contractility.    -   89. A method preventing and/or treating of a cardiac dysfunction        due, at least in part, to a cardiac insult, the method        comprising administering the compound of any one of items 1 to        82 or a pharmaceutically acceptable salt thereof to a subject in        need thereof.    -   90. The method of item 89, wherein the cardiac dysfunction is a        chronic condition such as heart failure, for example congestive        heart failure, particularly when drug-induced as well as acute        conditions such as a myocardial infarction.    -   91. The method of item 89 or 90, wherein the cardiac dysfunction        is coronary artery disease, heart attack, hypertension, faulty        heart valves, cardiomyopathy, myocarditis, congenital heart        defects, diabetes, or use of a cardiotoxic drug.    -   92. A method preventing and/or reducing cardiotoxicity        associated with use of a cardiotoxic drug, and/or reversing the        cardiotoxic effects thereof, the method comprising administering        the compound of any one of items 1 to 82 or a pharmaceutically        acceptable salt thereof to a subject in need thereof.    -   93. The method of item 92, wherein the cardiotoxic drug is        doxorubicin or imatinib.    -   94. The method of item 93, wherein the cardiotoxic drug is        doxorubicin.

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

FIG. 1 is a scheme showing the typical progression ofchemotherapy-induced heart failure;

FIG. 2 shows the general synthetic procedure for producing the compoundsof the invention;

FIG. 3 shows the dose-response for cardiomyocyte monologues treated withDoxorubicin (DOX) (300 nM) and/or with LCB2122 for 6 hours (data shownare the mean±SEM of N=3 per group);

FIG. 4 shows the dose-response for cardiomyocyte monologues treated withDoxorubicin (DOX) (300 nM) and with lower concentrations of LCB2122 orwith the vehicle;

FIG. 5 shows the time course for cardiomyocyte monologues treated withDoxorubicin (DOX) (300 nM) and/or with LCB2122;

FIG. 6 shows the dose-response for cardiomyocyte monologues treated withImatinib (Imat) (5 nM) and/or with LCB2122 for 6 hours;

FIG. 7 is a micrograph showing actinin stained cardiomyocytes treatedwith a) a vehicle (DMSO) or b) with LCB2122;

FIG. 8 is a plot representing the percent growth inhibition as measuredby Cell Titer Glo assays done on HepG2 cells treated with varyingconcentrations of DOX+/−LCB-2122 (1 M);

FIG. 9 shows the ejection fraction measured by in vivo echocardiographybefore and after treatment with DOX (5 mg/kg) in the presence andabsence of LCB2122;

FIG. 10 shows the left ventricle posterior wall thickness measured by invivo echocardiography before and after treatment with DOX (5 mg/kg) inthe presence and absence of LCB2122;

FIG. 11 shows the % of apoptotic nuclei as measured by TUNEL positiveassay after treatment with DOX (5 mg/kg) in the presence and absence ofLCB2122;

FIG. 12 shows the ejection fraction as measured by echocardiography invehicle and LCB2122 treated (2.0 μg/kg/day for 4 weeks wild-type (WT) orAT1R mice (HFmice) pre- and 4 weeks post-minipumps (The data shown areindividual values of each model of human HF); and

FIG. 13 shows a) ANF transcript changes using QPCR analysis on reversetranscribed RNA from vehicle and LCB2122 (2.0 μg/Kg/day, 4 weeks)treated WT and AT1R mice ventricles and b) quantification of an ANFELISA assay done on blood plasma samples from vehicle and LCB2122 (2.0μg/Kg/day, 4 weeks) treated WT and AT1R mice ventricles.

DETAILED DESCRIPTION OF THE INVENTION

An object of this invention is the identification of novel nucleosideand nucleotide analogues that can be used as cardioprotective agents.The invention thus relates to compounds useful as cardioprotectiveagents and to pharmaceutical compositions comprising these compounds.

The nucleoside and nucleotide analogues of the invention comprisetetrahydrofuranyl or tetrahydrothienyl moieties with a quaternarystereogenic all-carbon center at the 2′ position and a phosphonate esterat C5′. Pharmaceutically acceptable salts of these compounds are alsopart of the invention.

The invention provides compounds of formula:

or a pharmaceutically acceptable salt thereof,

wherein:

-   -   A and B are C₁-C₆ alkyl, mono- to per-halo C₁-C₆ alkyl,        —(CH₂)_(n)M, —C≡N, or

-   -    with the proviso that:        -   A is different from B,        -   when one of A and B is methyl, the other is not —CF₃, and        -   when one of A and B is C₂-C₆ alkyl, the other is not C₂-C₆            fluoroalkyl;    -   n is 1 to 3;    -   R₁ is

-   -   R₂ is the same or different, preferably the same, and is C₁-C₆        alkyl;    -   M is —OR₃, —SR₃, aryl, —C(O)OR₃, or —OC(O)R₄;    -   R₃ is —H, C₁-C₆ alkyl, aryl, aryl-C₁-C₆ alkyl, C₁-C₆ alkylaryl,        wherein each of the alkyl and aryl groups is optionally        substituted with one or more groups selected from halo, mono- to        per-halo C₁-C₆ alkyl, —CN, —C(O)OH, —C(O)OR₄, —N₃, —C₁-C₆        alkyl-C(O)OR₄, —CF₃, —C₁-C₆ alkyl-N₃, and —SiF₅;    -   R₄ is C₁-C₆ alkyl, aryl, heteroaryl, C₁-C₆ alkylaryl, aryl-C₁-C₆        alkyl, wherein each of the alkyl, aryl and heteroaryl groups is        optionally substituted with one or more groups selected from        halo, —CN, alkynyl, alkynyloxy, —C(O)OH, —N₃, —CF₃, —C₁-C₆        alkyl-N₃, —SiF₅, —NH₂, and —NHR₃;    -   C and D are independently —H, halo, azido, —OR₃, —CN, or —CF₃;    -   X is O or S; and    -   Base is:

-   -   R₅ is —H, —C(O)—C₁-C₄ alkyl, aryl, alkylaryl, or arylalkyl,        wherein each of the alkyl and aryl group is optionally        substituted with one or more groups selected from halo, —R₄,        —CF₃, and —N₃.

In embodiments, the compound is of the formulae:

preferably of formulae:

and more preferably of formulae:

In embodiments R₁ are

wherein R₂ is methyl, ethyl, isopropyl, butyl, tert-butyl as describedabove. In more preferred embodiments, R₁ is —CH₂OP(O)(OR₂)₂. In yet morepreferred embodiments R₁ is —CH₂OP(O)(OEt)₂.

In embodiments, A and B are C₁-C₆ alkyl, —(CH₂)_(n)M, —C≡N, or

preferably C₁-C₆ alkyl or —(CH₂)_(n)M, wherein n and M are as definedabove. In preferred embodiments, one of A or B is C₁-C₆ alkyl while theother is —(CH₂)_(n)M, —C≡N, or

preferably —(CH₂)_(n)M. In more preferred embodiments, A is C₁-C₆ alkyland B is —(CH₂)_(n)M, —C≡N, or

preferably —(CH₂)_(n)M. In yet more preferred embodiments:

the C₁-C₆ alkyl is methyl,

n is 1,

M is —OR₃ or —O(CO)R₄, preferably —OR₃, R₃ and R₄ being as definedabove.

In yet more preferred embodiments, R₃ in M of A/B is —H, C₁-C₆ alkyl, oraryl-C₁-C₆ alkyl, the aryl of the aryl-C₁-C₆ alkyl being optionallysubstituted with one or more:

-   -   halo, preferably —F,    -   mono- to per-halo C₁-C₆ alkyl, preferably per-halo C₁-C₆ alkyl,        the alkyl preferably being methyl, the halo preferably being —F,        more preferably —CF₃,    -   —N₃, and/or    -   —C₁-C₆ alkyl-N₃, preferably —CH₂—N₃.        In embodiments, the aryl-C₁-C₆ alkyl is benzyl optionally        substituted as noted above. In embodiments, the alkyl is methyl        or propyl. In more preferred embodiments, R₃ is —H, methyl,        isopropyl, benzyl,

preferably —H or benzyl, and more preferably —H.

In yet more preferred embodiments, R₄ in M of A/B is aryl or heteroaryloptionally substituted as noted above, preferably benzoylphenyl

or indole-5-carbonylpheny;

both of which optimally and preferably substituted with alkynyl(preferably prop-2-yn-1-yl (—CH₂—C≡CH)) or alkynyloxy (preferablyprop-2-yn-1-yloxy (—O—CH₂—C≡CH)), wherein the aryl is preferablysubstituted with alkynyloxy and the heteroaryl is preferably substitutedwith alkynyl. More preferably, R₃ is

In embodiments, C and D are independently —H, halo (preferably —F), —OR₃(preferably —OH), —CN and CF₃, preferably —H, halo, or —OR₃ (preferably—OH). In preferred embodiments, one of C or D is —H and the other ishalo or —OR₃ (preferably the “other” is —OR₃, which is preferably —OH).In more preferred embodiments, C is —H and D is halo or —OR₃ (preferablyD is —OR₃, which is preferably —OH). In yet more preferred embodiments,C is H and D is —OR₃ (preferably —OH).

In embodiments, X is O.

In embodiments, R₅ represents —H, —C(O)—C₁-C₄ alkyl, arylalkyl, or aryl,wherein each of the aryl groups is optionally substituted with one ormore groups selected from halo, —R₄, —CF₃, and —N₃, preferably halo and—R₄. In embodiments, R₅ represents —H. In embodiments, R₅ represents—C(O)—C₁-C₄ alkyl. In embodiments, the alkyl group in —C(O)—C₁-C₄ alkylin R₅ is propyl. In embodiments, R₅ represents arylalkyl. Inembodiments, the aryl group of the arylalkyl in R₅ is optionallysubstituted with one or more —R₄. In other embodiments, the aryl groupof the arylalkyl in R₅ is unsubstituted. In embodiments, the arylalkylin R₅ is benzyl. In embodiments, R₅ represents aryl. In embodiments, thearyl in R₅ is optionally substituted with one or more, preferably one, For —CF₃, preferably —CF₃. In embodiments, the aryl in R₅ is phenyl. Inpreferred embodiments, R₅ represents —H, —C(O)— propyl, benzyl, orp-trifluoromethylphenyl.

In preferred embodiments, base is:

preferably base is:

more preferably base is:

In embodiments, base is:

In preferred embodiments, base is

In preferred embodiments, base is

In preferred embodiments, base is

In preferred embodiments, base is

In preferred embodiments, base is

In preferred embodiments, base is

In preferred embodiments, base is

In such embodiments, R₅ is preferably arylalkyl, wherein the aryl groupis optionally substituted with one or more groups selected from halo,—R₄, —CF₃, and —N₃, preferably —R4. In preferred embodiments, R₅ isbenzyl optionally substituted with —R₄ and more preferably R₅ isunsubstituted benzyl.

In preferred embodiments, base is

In preferred embodiments, base is

In such embodiments, R₅ preferably represents —C(O)—C₁-C₄ alkyl or aryloptionally substituted with one or more groups selected from halo, —R₄,—CF₃, and —N₃. In embodiments, R₅ represents aryl optionally substitutedwith one or more groups selected from halo, —R₄, —CF₃, and —N₃,preferably —CF₃. In preferably embodiments, R₅ represents phenyloptionally substituted with —CF₃, and most preferablyp-trifluoromethylphenyl. In embodiments, R₅ represents —C(O)—C₁-C₄ alkyland most preferably —C(O)-propyl. In preferred embodiments, base is

As noted above, R₁ is

R₂ being as defined above. In embodiments, R₁ is

In alternative embodiments, R₁ is

In embodiments, R₂ is methyl, ethyl, isopropyl or tert-butyl, preferablyethyl or isopropyl, more preferably ethyl. In a preferred embodiment, R₁represents

Preferred compounds of the invention include:

or a pharmaceutically acceptable salt thereof to the subject.

More preferred compounds of the invention include:

or a pharmaceutically acceptable salt thereof.

Even more preferred compounds of the invention include:

and yet more preferred compounds include:

most preferably

As noted above, “pharmaceutically acceptable salts” of the compoundsdescribed herein are included within the scope of the present invention.Such salts may be prepared from pharmaceutically acceptable non-toxicbases including inorganic bases and organic bases. Salts derived frominorganic bases include sodium, potassium, lithium, ammonium, calcium,magnesium, ferrous, zinc, copper, manganous, aluminum, ferric, manganicsalts and the like. Particularly preferred are the potassium, sodium,calcium and magnesium salts. Salts derived from pharmaceuticallyacceptable organic non-toxic bases include salts or primary, secondary,and tertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines and basic ion exchange resin, such asisopropylamine, tri-methylamine, diethanolamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylamino-ethanol, tometheamine, lysine, arginine, histidine,caffeine, procaine, hydrabamine, choline, imidazole, betaine,ethylenediamine, glucosamine, methylglucamine, theobromine, purinespiperazine, N,N-dibenzylethylenediamine, piperidine, N-ethyl-piperidine,morpholine, N-ethylmorpholine, polyamine resins and the like.“Pharmaceutically acceptable salts” also refers to those salts thatretain the biological effectiveness of the free bases and that are notbiologically or otherwise undesirable, formed with inorganic acids suchas hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like, as well as organic acids such as aceticacid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like. See, for example, S. M. Berge, et al.,“Pharmaceutical Salts,” J. Pharm. Sci., 1977; 66:1-19 which isincorporated herein by reference.¹²

Pharmaceutical Compositions

The present invention also relates to pharmaceutical compositionscomprising a pharmaceutically acceptable carrier, excipient or diluentand a compound of the invention as defined above or a pharmaceuticallyacceptable salt thereof.

Administration of the compounds of the invention, or theirpharmaceutically acceptable salts, in pure form or in an appropriatepharmaceutical composition, can be carried out via any of the acceptedmodes of administration or agents for serving similar utilities. Thus,administration can be, for example, orally, nasally, parenterally(intravenous, intramuscular, or subcutaneous), topically, transdermally,intravaginally, intravesically, intracistemally, or rectally, in theform of solid, semi-solid, lyophilized powder, or liquid dosage forms,such as, for example, tablets, suppositories, pills, soft elastic andhard gelatin capsules, powders, solutions, suspensions, or erosols, orthe like, preferably in unit dosage forms suitable for simpleadministration of precise dosages. In embodiments, administration maypreferably be by the oral route.

The compositions of the invention include a conventional pharmaceuticalcarrier or excipient and a compound of the invention as the/an activeagent, and, in addition, may include other medicinal agents,pharmaceutical agents, carriers, adjuvants, etc. In particular,compositions of the invention may be used in combination with anticanceror other agents that are generally administered to a patient beingtreated for cancer. Adjuvants include preserving, wetting, suspending,sweetening, flavoring, perfuming, emulsifying, and dispensing agents.

Prevention of the action of microorganisms can be ensured by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, and the like. It may also bedesirable to include isotonic agents, for example sugars, sodiumchloride, and the like. Prolonged absorption of injectablepharmaceutical forms can be brought about using agents delayingabsorption, for example, aluminium monostearate and gelatin.

If desired, a pharmaceutical composition of the invention may alsocontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents, antioxidants, and the like,such as, for example, citric acid, sorbitan monolaurate, triethanolamineoleate, butylated hydroxytoluene, etc.

Compositions suitable for parenteral injection may comprisephysiologically acceptable sterile aqueous or non-aqueous solutions,dispersions, suspensions or emulsions, and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and non-aqueous carriers, diluents,solvents or vehicles include water, ethanol, polyols (propyleneglycol,polyethyleneglycol, glycerol, and the like), suitable mixtures thereof,vegetable oils (such as olive oil) and injectable organic esters such asethyl oleate. Proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersions and by the use of surfactants.

One preferable route of administration is oral, using a convenient dailydosage regimen that can be adjusted according to the degree of severityof the disease-state to be treated.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is admixed with at least one inert customary excipient (orcarrier) such as sodium citrate or dicalcium phosphate or (a) fillers orextenders, as for example, starches, lactose, sucrose, glucose,mannitol, and silicic acid, (b) binders, as for example, cellulosederivatives, starch, alignates, gelatin, polyvinylpyrrolidone, sucrose,and gum acacia, (c) humectants, as for example, glycerol, (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, croscarmellose sodium, complexsilicates, and sodium carbonate, (e) solution retarders, as for exampleparaffin, (f) absorption accelerators, as for example, quaternaryammonium compounds, (g) wetting agents, as for example, cetyl alcohol,and glycerol monostearate, magnesium stearate and the like (h)adsorbents, as for example, kaolin and bentonite, and (i) lubricants, asfor example, talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In thecase of capsules, tablets, and pills, the dosage forms may also comprisebuffering agents.

Solid dosage forms, as described above can be prepared with coatings andshells, such as enteric coatings and others well known in the art. Theymay contain pacifying agents, and can also be of such composition thatthey release the active compound or compounds in a certain part of theintestinal tract in a delayed manner. Examples of embedded compositionsthat can be used are polymeric substances and waxes. The activecompounds can also be in microencapsulated form, if appropriate, withone or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Suchdosage forms are prepared, for example, by dissolving, dispersing, etc.,a compound(s) of the invention, or a pharmaceutically acceptable saltthereof, and optional pharmaceutical adjuvants in a carrier, such as,for example, water, saline, aqueous dextrose, glycerol, ethanol and thelike; solubilizing agents and emulsifiers, as for example, ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol,dimethylformamide; oils, in particular, cottonseed oil, groundnut oil,corn germ oil, olive oil, castor oil and sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters ofsorbitan; or mixtures of these substances, and the like, to thereby forma solution or suspension.

Suspensions, in addition to the active compounds, may contain suspendingagents, as for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, or mixtures of thesesubstances, and the like.

Compositions for rectal administrations are, for example, suppositoriesthat can be prepared by mixing the compounds of the present inventionwith for example suitable non-irritating excipients or carriers such ascocoa butter, polyethyleneglycol or a suppository wax, which are solidat ordinary temperatures but liquid at body temperature and therefore,melt while in a suitable body cavity and release the active componenttherein.

Dosage forms for topical administration of a compound of this inventioninclude ointments, powders, sprays, and inhalants. The active componentis admixed under sterile conditions with a physiologically acceptablecarrier and any preservatives, buffers, or propellants as may berequired. Ophthalmic formulations, eye ointments, powders, and solutionsare also contemplated as being within the scope of this invention.

Generally, depending on the intended mode of administration, thepharmaceutically acceptable compositions will contain about 1% to about99% by weight of a compound(s) of the invention, or a pharmaceuticallyacceptable salt thereof, and 99% to 1% by weight of a suitablepharmaceutical excipient. In one example, the composition will bebetween about 5% and about 75% by weight of a compound(s) of theinvention, or a pharmaceutically acceptable salt thereof, with the restbeing suitable pharmaceutical excipients.

Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art; for example, see Remington'sPharmaceutical Sciences, 18^(th) Ed., (Mack Publishing Company, Easton,Pa., 1990).¹³ The composition to be administered will, in any event,contain a therapeutically effective amount of a compound of theinvention, or a pharmaceutically acceptable salt thereof, for treatmentof a disease-state in accordance with the teachings of this invention.

The compounds of the invention, or their pharmaceutically acceptablesalts, are administered in a therapeutically effective amount which willvary depending upon a variety of factors including the activity of thespecific compound employed, the metabolic stability and length of actionof the compound, the age, body weight, general health, sex, diet, modeand time of administration, rate of excretion, drug combination, theseverity of the particular disease-states, and the host undergoingtherapy. The compounds of the present invention can be administered to apatient at dosage levels in the range of about 0.1 to about 1,000 mg perday. For a normal human adult having a body weight of about 70kilograms, a dosage in the range of about 0.01 to about 100 mg perkilogram of body weight per day is an example. The specific dosage used,however, can vary. For example, the dosage can depend on a number offactors including the requirements of the patient, the severity of thecondition being treated, and the pharmacological activity of thecompound being used. The determination of optimum dosages for aparticular patient is well known to one of ordinary skill in the art.

Use of the Compounds Cardioprotection

The present invention relates to the use of the above compounds and theabove pharmaceutical composition for providing cardioprotection in asubject in need thereof. In other words, the invention relates to amethod of providing cardioprotection in a subject in need thereof, themethod comprising administering the compound to the subject.

Indeed, as shown in the examples below, the compounds of the inventionreduced abnormal cardiomyocyte apoptosis, loss of ejection fractionand/or loss of ventricular wall thickness induced by cardiotoxicanticancer drugs. They also significantly improved the cardiac functionin AT1R mice, which are a model for heart failure.

Herein, “cardioprotection” means preserving, reducing deterioration of,and/or improving cardiac function of a heart that has been subjected, issubjected, and/or will be subjected to a cardiac insult. The abovesubject is thus a subject whose heart has been subjected, is subjected,or will be subjected to one or more of cardiac insults.

The subject is thus at risk of developing or presents a deterioration ofcardiac function due, at least in part, to the cardiac insult. Inembodiments, the cardiac function may be the ejection fraction of theheart or the cardiac contractility.

Cardioprotection includes preventing, reducing, and/or reversing heartdamage due to the cardiac insult. Non-limiting examples of heart damagethat can be prevented, reduced or reversed include abnormalcardiomyocyte apoptosis, cardiac remodeling including changes (loss orgain) in heart wall thickness, decrease in ejection fraction especiallyresulting in inadequate cardiac output, poor organ perfusion, and/orloss of cardiac contractility.

Cardioprotection also includes the prevention and/or treatment of acardiac dysfunction due, at least in part, to the cardiac insult. Thus,in such embodiments, the above subject is a subject at risk ofdeveloping or presenting a cardiac dysfunction due, at least in part, tothe cardiac insult. Non-limiting examples of cardiac dysfunctionsinclude chronic conditions such as heart failure, for example congestiveheart failure, particularly when drug-induced, as well as acuteconditions such as a myocardial infarction.

Non-limiting examples of cardiac insults include:

coronary artery disease and heart attack;

hypertension;

faulty heart valves;

cardiomyopathy;

myocarditis;

congenital heart defects;

diabetes; and

the use of cardiotoxic drugs, particularly anticancer drugs.

Thus, in specific embodiments, cardioprotection include the preventionor reduction of cardiotoxicity associated with the use of a cardiotoxicdrug, and/or reversing the cardiotoxic effects thereof. It iscontemplated that the compound of the invention, may be used before,during or after a course of treatment with a cardiotoxic drug. Use ofthe compound of the invention “during” the course of treatment with acardiotoxic drug include concurrent, subsequent, or alternatingadministration of both drugs during the course of treatment with thecardiotoxic drug. In such embodiments, it is contemplated that theamount and/or frequency of therapy with such drugs could be increasedwithout a concomitant increase in cardiotoxicity.

Herein, a “cardiotoxic drug” is a drug that causes damage to the heart,and particularly in embodiments to the cardiomyocytes. Non-limitingexamples of cardiotoxic drugs include anticancer drugs such asanthracyclines, (including doxorubicin, epirubicin, daunorubicin,idarubicin, and mitoxantrone), monoclonal antibodies (includingtrastuzumab (TRZ), bevacizumab, cetuximab, brentuzimab, ipilimumab,panitumumab, pertuzumab, and rituximab) tyrosine kinase inhibitors(including imatinib, dasatinib, nilotinib, vermurafenib, sorafenib,sunitinib, erlotinib, gefitinib, lapatinib, and pazopanib), proteasomeinhibitors (including bortezomib, carfilzomib, tamoxifen, abiraterone,anastrozole, exemestane, letrozole, 5-fluorouracil, capecitabine,cisplatin, cyclophosphamide, and ifosfamide) and antimicrotubule agents(including paclitaxel, nab-paclitaxel, and docetaxel).^(3,14) Inpreferred embodiments, the cardiotoxic drug is doxorubicin or imatinib,preferably doxorubicin.

The present invention also relates to the use of the above compounds andthe above pharmaceutical composition for providing in vitrocardioprotection. In other words, the invention relates to a method ofproviding in vitro cardioprotection, the method comprising administeringthe compound to the heart. Cardioprotective effects for these moleculescould indeed be used in vitro for example to prevent damage caused byischemia during heart transplantation (cold ischemia reperfusion injury,IRI).¹⁵

General Synthetic Procedure

The compounds of the invention can be prepared using reagents readilyavailable. See the reaction scheme in FIG. 2 as well as the workingexamples provided below.

Definitions

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext.

The terms “comprising”, “having”, “including”, and “containing” are tobe construed as open-ended terms (i.e., meaning “including, but notlimited to”) unless otherwise noted.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All subsets of values within the ranges arealso incorporated into the specification as if they were individuallyrecited herein.

Similarly, herein a general chemical structure with various substituentsand various radicals enumerated for these substituents is intended toserve as a shorthand method of referring individually to each and everymolecule obtained by the combination of any of the radicals for any ofthe substituents. Each individual molecule is incorporated into thespecification as if it were individually recited herein. Further, allsubsets of molecules within the general chemical structures are alsoincorporated into the specification as if they were individually recitedherein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed.

No language in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Herein, the term “about” has its ordinary meaning. In embodiments, itmay mean plus or minus 10% or plus or minus 5% of the numerical valuequalified.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

Herein, the terms “alkyl”, “alkylene”, “alkenyl”, “alkenylene”,“alkynyl”, “alkynylene” and their derivatives (such as alkoxy,alkyleneoxy, etc.) have their ordinary meaning in the art. For morecertainty:

Term Definition alkyl monovalent saturated aliphatic hydrocarbon radicalof general formula —C_(n)H_(2n+1) alkenyl monovalent aliphatichydrocarbon radical similar to an alkyl, but comprising at least onedouble bond alkynyl monovalent aliphatic hydrocarbon radical similar toan alkyl, but comprising at least one triple bond alkyloxy or alkoxymonovalent radical of formula —O-alkyl alkynyloxy monovalent radical offormula —O-alkynyl

It is to be noted that, unless otherwise specified, the hydrocarbonchains of the above groups can be linear or branched. Further, unlessotherwise specified, these groups can contain between 1 and 18 carbonatoms, more specifically between 1 and 12 carbon atoms, between 1 and 6carbon atoms, between 1 and 3 carbon atoms, or contain 1 or 2,preferably 1, or preferably 2 carbon atoms.

Herein, the terms “cycloalkyl”, “aryl”, “heterocycloalkyl”, and“heteroaryl” have their ordinary meaning in the art. For more certainty:

Term Definition aryl a monovalent aromatic hydrocarbon radicalpresenting a delocalized conjugated Π system, most commonly anarrangement of alternating single and double bonds, between carbon atomsarranged in one or more rings, wherein the rings can be fused (i.e.share two ring atoms), for example:

or linked together through a covalent bond, for example:

or linked together through a radical that allow continuation of thedelocalized conjugated Π system between the rings (e.g. —C(═O)—, —NRR—),for example:

heteroaryl aryl wherein at least one of the ring carbon atoms isreplaced by a heteroatom, such as nitrogen or oxygen. Examples ofheteroaryl include:

cycloalkyl monovalent saturated aliphatic hydrocarbon radical of generalformula C_(n)H_(2n-1), wherein the carbon atoms are arranged in one ormore rings (also called cycles). heterocyclo- cycloalkyl wherein atleast one of the carbon atoms is alkyl replaced by a heteroatom.

It is to be noted that, unless otherwise specified, the ring(s) of theabove groups can each comprise between 4 and 8 ring atoms, preferablybetween 5 or 6 ring atoms. Also, unless otherwise specified, the abovegroups may preferably comprise one or more rings, preferably 1 or 2rings, more preferably a single ring.

Herein, the term “heteroatom” means nitrogen, oxygen, sulfur,phosphorus, preferably nitrogen or oxygen.

Herein, the term “arylalkyl” means an alkyl substituted with an aryl,the alkyl and aryl being as defined above. An arylalkyl groups attachesto the rest of a molecule via its alkyl moiety.

Herein, the term “alkylaryl” means an aryl substituted with an alkyl,the alkyl and aryl being as defined above. An alkylaryl groups attachesto the rest of a molecule via its aryl moiety.

Herein, “halo” refers to halogen atoms, which include fluorine (F),chlorine (Cl), bromine (Br), and iodine (I).

Herein, “azido” refers to a radical of formula N₃, i.e. —N═N⁺═N⁻, whichis in resonance with —N⁻—N⁺≡N.

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following non-restrictivedescription of specific embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention is illustrated in further details by the followingnon-limiting examples.

Example 1—Cardioprotective Activity In Vitro Experiments

In vitro experiments were carried out to test the direct effect ofcompounds of the invention LCB2122, LC2165, and LCB2191 oncardiomyocytes.

Primary cardiomyocyte monologues covered by serum free media (SF) weretreated with DMSO (vehicle), Doxorubicin (DOX) (300 nM), Imatinib (Imat)(5 μM), and/or LCB2122 at varying concentrations (0.1, 0.5, 1.5 at 10μM) diluted in SF for 3, 6, 18 or 24 hours. The TUNEL assay (TerminalDeoxynucleotidyltransferase mediated dUTP End Labeling) was utilized todetect apoptotic nuclei using an Apop Tag Red in Situ ApoptosisDetection Kit (Millipore, Temecula, Calif.). A Zeis fluorescentmicroscope was used for image acquisition. Cell counting was done usingthe Imager® Software.

As seen in FIGS. 3 and 4, LCB2122 completely prevented DOX-inducedapoptotic cell death with an EC₅₀ of 500 nM (FIG. 4). LCB2122 did notinduce apoptosis in these cells at any time points (FIG. 5). DOX-inducedapoptosis was however blocked by LCB2122 at 3, 6 or 24 hours.

As seen in FIG. 6, the apoptosis induced by Imatinib was also preventedby LCB2122 with an EC₅₀ of 5 μM.

The cardioprotective profile of LCB2122, LC2165, LCB2191 and LCB2177were also investigated. As above, primary cardiomyocytes were treatedwith 3 μM of Doxorubicin for 6 hours alone or with the above compoundsof the invention. The TUNEL assay was utilized to detect apoptoticnuclei. The results are shown in Table 1. LCB2122 showed an IC₅₀ of 1 μMin this assay. LCB2165, LCB2191 and LCB2177 were also active with IC₅₀'sof 5 μM.

TABLE 1 Compound IC₅₀ LCB2122 1 μM LCB2165 5 μM LCB2191 5 μM LCB2177 5μM

Immunofluorescence were performed on cardiomyocytes as previouslydescribed,¹⁶ using a sarcomeric alpha-Actinin antibody (1/500).

FIG. 7 is a micrograph showing α-actinin immunostaining (straight lines)of 2% PFA-fixed cardiomyocytes treated with a) vehicle (DMSO) or b)LCB2122 (5 uM) for six hours. Note how LCB2122 does not altercytoskeletal organization or cell size. White spots mark the nuclei.

Furthermore, it was observed (FIG. 8) that LCB2122 did not affect theantiproliferative effect of DOX. Indeed, the IC₅₀ of DOX in HepG2 humancancer cell line was identical in the absence or presence of LCB2122.FIG. 8 is a plot representing the percent growth inhibition as measuredby Cell Titer Glo assays done on HepG2 cells treated with varyingconcentrations of DOX+/−LCB-2122 (1 μM). With up to 4 μM, LCB2122 had noeffect on HepG2 sensitivity to DOX.

In Vivo Experiments

Mice were handled in accordance with institutional guidelines for animalcare. Experiments were approved by the institutional Animal CareCommittees and the investigation conforms with the Guide for the Careand Use of Laboratory Animals published by the US National Institutes ofHealth (NIH Publication N. 85-23, revised 1985). Doxorubicin treatmentwas a single ip injection of 5 or 15 mg/kg as previously described byAries et al.⁶ ALZET micro-osmotic pumps (Model 1002, 0.25 μl/hr, 14days) were filled with DMSO, Ang II (0.5 μgKg/day) LCB2122 (2.0 or 2.8μg/Kg/day) diluted in normal saline and inserted subcutaneously in micefor two weeks. M-mode echocardiography was performed using Visual-SonicsVEVO 2100 system and 30-MHz linear array transducer, on lightlyanesthetized mice using 20% isofluorane, 80 ml/min of 100% oxygen, asdescribed by Aries et al. and echocardiographic indices were calculatedas described by Yang et al.¹⁷ Heart failure was defined as EF<45%.

As seen in FIG. 9, the ejection fraction (EF, %), which is an index ofcardiac contractility, was decreased significantly in mice after twoweeks of DOX ip injection. LCB2122 treated mice (micropump for twoweeks) were protected against DOX-induced toxicity (i.e. EF decrease).

DOX (5 μg/kg) induced also a loss of ventricular wall thickness in wildtype mice. The LCB2122 treated mice were protected against thisDOX-induced wall thickness (see FIG. 10).

After these 2 weeks, heart cardiomyocytes were also tested by TUNELassay (on heart sections). As seen in FIG. 11, a significant decrease ofapoptotic cardiomyocytes was noted when LCB2122 was used.

Transgenic mice overexpressing the human angiotensin II type 1 receptor⁷(AT1R) under the control of the mouse α-myosin heavy chain weregenerated. These mice are a model of human HF. Cardiomyocyte specificoverexpression induced in basal conditions overtime, morphologic changesthat mimic those observed during the development of cardiac hypertrophyin humans. In order words, these mice develop age-dependent HF. Thesemice indeed displayed remodeling with increased expression ofventricular clinical natriuretic factor and interstitial collagendeposition and died prematurely of heart failure.

The effect of LCB2122 was studied in AT1R mice and wild-type mice.

As seen in FIG. 12, echocardiography shows changes in heart ejectionfraction (EF %) for 60 day old AT1R transgenic mice. All AT1R mice hadEF % less than 40%, similar to HF patients in human. Ejection fraction(%) was decreased significantly in mice after 4 weeks in 60 day AT1Rmice. LCB2122 treated AT1R mice (2.0 μg/kg/day for 4 weeks) had theircardiac functions improved as opposed to untreated mice. In fact, thedata shows a significant improvement of the cardiac function for AT1Rmice treated for 4 weeks (2.0 μg/kg per day) with LCB2122. LCB2122 didnot alter cardiac function of normal mice. The data shown are theindividual values of each mouse at weeks 0 and 4. All WT mice had normalEF % independent of LCB2122 treatment throughout the study. All AT1Rmice were in HF prior to any treatment. The EF % of AT1R mice+LCB2122continued to go up, and after four weeks 55% of these mice had an EF %equal and above 40%.

As seen in FIG. 13, both levels of atrial natriuretic factor (ANF) inRNA or ANF measured by ELISA were attenuated after the treatment asopposed to the ventricles of treated mice.

Indeed, FIG. 13 a) shows ANF transcript changes using QPCR analysis onreverse transcribed RNA from vehicle and LCB2122 (2.0 μg/Kg/day, 4weeks) treated WT and AT1R mice ventricles. The massive ANF upregulationseen in AT1R mice usually indicative of cardiac stress is attenuated inLCB2122 treated AT1R mice.

FIG. 13 b) quantification of an ANF ELISA assay done on blood plasmasamples from vehicle and LCB2122 (2.0 μg/Kg/day, 4 weeks) treated WT andAT1R mice ventricles. Once again, the increase in ANF plasma levels wasattenuated in the LCB2122 treated AT1R mice.

Example 2—Chemical Synthesis Example 2.1—Intermediate Compound

(4R,5R)-3-bromo-4-hydroxy-5-(hydroxymethyl)-3-methyldihydrofuran-2(3H)-one

To a solution of precooled glyceraldehyde (24 g, 185 mmol) in dryacetonitrile (710 mL) at −10° C. under Ar, MgBr₂.OEt₂ (37 g, 142 mmol)is added. After 15 minutes, all the solids were in solution and neatenolate (50.7 g, 142 mmol) precooled at −20° C. was added via cannuladuring 10 minutes. The mixture was stirred for 23 h at 0° C., andquenched by addition of 200 mL ice-H₂O at 0° C. The mixture was dilutedwith ethyl acetate, washed 2×200 mL with distilled water, the organicphase was dried over MgSO₄, and concentrated to produce clear brown oil(50.7 g), which was used for the next step. HCl conc (10 mL, 121.8 mmol)was added dropwise to a solution of aldol adducts (50.7 g, 137 mmol) inTHF (275 mL) at 0° C. and open atmosphere for 20 minutes. After 50minutes, the reaction was warmed to room temperature. After 5 h, thereaction mixture was concentrated producing dark green oil that waspassed trough a bed of SiO₂ (200 mL) and rinsed with a mixture ofCH₂Cl₂/EtOAc 50%. The dark brown solid was washed with hexanes, thentwice with Hexanes/EtOAc (95:5) producing a clear brown solid (19 g, 59%over 2 steps).

A: major lactone (3,4-anti). ¹H NMR (500 MHz, Methanol-d₄) δ 4.18 (ddd,J=8.4, 4.2, 2.1 Hz, 1H), 3.97 (dd, J=13.0, 2.1 Hz, 1H), 3.82 (d, J=8.4Hz, 1H), 3.73 (dd, J=13.0, 4.2 Hz, 1H), 1.86 (s, 3H). ¹³C NMR (126 MHz,CD₃OD) δ 174.4, 84.3, 74.3, 62.4, 59.9, 24.4.

B: minor lactone (3,4-anti) ¹H NMR (500 MHz, Methanol-d₄) δ 4.64 (d,J=6.3 Hz, 1H), 4.24 (ddd, J=6.3, 5.0, 3.2 Hz, 1H), 3.96-3.84 (m, 2H),3.82-3.75 (m, 1H), 1.82 (s, 3H). ¹³C NMR (126 MHz, CD₃OD) δ 175.7, 86.1,78.0, 61.4, 58.8, 22.3. R_(f)=0.05 (30% ethyl acetate in hexanes).

Example 2.2—Intermediate Compound

(4R,5R)-3-bromo-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxy-3-methyldihydrofuran-2(3H)-one

TBSCl (12.7 g, 84.4 mmol) was added to a mixture of lactones (19 g, 84.4mmol), and imidazole (23 g, 337.7 mmol) in dry DMF (420 mL) under Ar at−40° C. The reaction was followed by TLC, and after 5 h, 0.06 equiv ofTBSCl (0.77 g, 5.10 mmol) was added. After 7 h in total, the reactionmixture was diluted with ethyl acetate (800 mL), washed sequentiallywith citric acid (0.1M, 400 mL), distilled water, brine, dried overMgSO₄ and concentrated to produce a brown oil (27 g, 94% yield).R_(f)=0.37 (30% ethyl acetate in hexanes); IR (neat) 3457, 2952, 2931,2855, 1771, 1256, 1132 cm⁻¹; Formula C₁₂H₂₃BrO₄Si; MW 339.2981; Formajor diastereomer from 3,4-anti aldol adduct: ¹H NMR (500 MHz, CDCl₃) δ4.13 (dt, J=8.1, 2.8 Hz, 1H), 4.01 (dd, J=12.1, 2.5 Hz, 1H), 3.94-3.83(m, 2H), 1.94 (s, 3H), 0.95-0.82 (m, 9H), 0.08 (d, J=7.8 Hz, 6H); ¹³CNMR (125 MHz, CDCl₃) δ 171.5, 82.7, 74.0, 62.3, 60.0, 26.0, 24.4, 18.4,−5.2, −5.3; For minor diastereomer from 3,4-anti aldol adduct: ¹H NMR(500 MHz, CDCl₃) δ 4.82 (dd, J=6.6, 2.5 Hz, 1H), 4.22 (ddd, J=6.4, 5.3,3.8 Hz, 1H), 4.00-3.88 (m, 2H), 1.88 (s, 3H), 0.90 (s, 9H), 0.10 (s,6H); ¹³C NMR (125 MHz, CDCl₃) δ 173.1, 83.0, 78.1, 61.7, 57.0, 26.0,22.1, 18.4, −5.2, −5.2; MS (ESI) m/z 361.0 (M+Na⁺, 100); HRMS calcd for[M+H⁺]: 339.0627, found: 339.0621; calcd for [M+Na⁺]: 361.0447, found:361.0442.

Example 2.3—Intermediate Compound

(4R,5R)-3-bromo-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-((dimethyl(vinyl)silyl)oxy)-3-methyldihydrofuran-2(3H)-one

Chlorodimethylvinylsilane (11.2 g, 13.2 mL, 92.9 mmol) was added to amixture of TBS-lactones (84.43 mmol) and dry pyridine (16.6 g, 17.0 mL,211.1 mmol) in dry CH₂Cl₂ (422 mL) under Ar at 0° C. The mixture wasbrought to room temperature slowly and after 23 h, 0.05 equiv ofchlorodimethylvinylsilane was added (509 mg, 0.6 mL). After 28 h intotal, the reaction mixture was concentrated, suspended in a mixture of20% ethyl acetate in hexanes, passed trough a pad of SiO₂, rinsed with500 mL of (20% ethyl acetate in hexanes), and concentrated to produceyellow oil (32.5 g, 96% yield). R_(f)=0.6 (30% ethyl acetate inhexanes); IR (neat) 2952, 2925, 2850, 1787, 1256, 1138 cm⁻¹; FormulaC₁₆H₃₁BrO₄Si₂; MW 423.4899; For major diastereomer from 3,4-anti aldoladduct:

¹H NMR (500 MHz, CDCl₃) δ 6.20-6.05 (m, 2H), 5.86 (dd, J=19.4, 4.6 Hz,1H), 4.16 (dt, J=7.9, 1.9 Hz, 1H), 4.03-3.98 (m, 2H), 3.78 (dd, J=12.6,2.0 Hz, 1H), 1.84 (s, 3H), 0.87 (s, 9H), 0.28 (s, 6H), 0.07 (d, J=10.4Hz, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 172.4, 136.4, 134.9, 82.5, 73.4,60.1, 58.6, 25.9, 24.9, 18.3, −1.3, −1.5, −5.2, −5.3;

For minor diastereomer from 3,4-anti aldol adduct:

¹H NMR (500 MHz, CDCl₃) δ 6.20-6.05 (m, 2H), 5.83 (dd, J=19.9, 4.1 Hz,1H), 4.84 (d, J=6.0 Hz, 1H), 4.13 (ddd, J=6.0, 4.0, 3.2 Hz, 1H), 3.96(dd, J=12.0, 3.8 Hz, 1H), 3.81 (dd, J=11.9, 3.6 Hz, 1H), 1.80 (s, 3H),0.90 (s, 9H), 0.30-0.28 (m, 6H), 0.08 (s, 6H); ¹³C NMR (125 MHz, CDCl₃)δ 173.5, 136.3, 134.7, 84.2, 77.4, 60.5, 57.5, 25.9, 22.5, 18.4, −1.4,−1.5, −5.2, −5.3; MS (ESI) m/z 447.1 (M+Na⁺, 100), 445.1 (M+Na⁺, 100),431.2 (83); HRMS calcd for [M+H⁺]: 423.1023, found: 423.1009; [M+NH₄ ⁺]:440.1288, found: 440.1274; calcd for [M+Na⁺]: 445.0842, found: 445.0831.

Example 2.4—Intermediate Compound

(3R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-3-methyl-3-vinyldihydrofuran-2(3H)-one

Et₃B (76.8 mL, 76.8 mmol, 1M in hexanes) was added via syringe with arate of 15.4 mL/h to a solution of lactones (32.5 g, 76.8 mmol) in drytoluene (153 mL) at 0° C., open atmosphere and vigorous stirring. After7 h, 0.1 equiv of BEt₃ (7.7 mL, 7.7 mmol, 1M in hexanes) was added. Thereaction was quenched after 8 h in total by consecutive addition ofmethanol (153 mL) and acetic acid (9.2 g, 8.8 mL, 153.6 mmol) at 0° C.and allowed to reach room temperature slowly (overnight). The reactionmixture was concentrated after 17 h and the resulting brown oil waswashed with hexanes (×1), passed through a pad of SiO₂ and rinsed with agradient of 50% ethyl acetate/hexanes to 100% ethyl acetate. The brownsolid was dissolved in CH₂Cl₂ and the title product was formed as awhite solid (7.08 g, 53% yield, only one diastereomer). The remainingbrown oil (8.52 g) was purified by silica gel column chromatography (30%ethyl acetate in hexanes) to yield the title product as a beige solid(4.68 g, 35%, 5:1 mixture of diastereomers). Overall yield 88% and ca.14:1 (3,4-anti:3,4-syn). R_(f)=0.09 (30% ethyl acetate in hexanes); IR(neat) cm⁻¹ 3419, 2936, 1766, 1100, 1041; Formula C₈H₁₂O₄; MW 172.1785;¹H NMR (500 MHz, CDCl₃) δ 6.07-5.92 (m, 1H), 5.27 (d, J=10.7 Hz, 1H),5.13 (d, J=17.7 Hz, 1H), 4.12-4.03 (m, 2H), 3.97-3.89 (m, 1H), 3.68 (dd,J=13.1, 3.8 Hz, 1H), 1.32 (s, 3H); ¹³C NMR (125 MHz, CD₃OD) δ 178.9,135.7, 117.0, 83.6, 75.8, 60.8, 52.7, 20.9; MS (ESI) m/z 195.1 (M+Na⁺,100), 173.1 (M+H⁺, 7); HRMS calcd for [M+H⁺]: 173.0814, found: 173.0804;calcd for [M+Na⁺]: 195.0633, found: 195.0625; [α]_(D) +53 (c 1.4,CH₃OH).

Example 2.5—Intermediate Compound

((2R,3S,4R)-3-(benzoyloxy)-4-methyl-5-oxo-4-vinyltetrahydrofuran-2-yl)methylbenzoate

Benzoyl chloride (26.9 g, 22.2 mL, 191.14 mmol) was added slowly to amixture of lactone (10.97 g, 63.71 mmol), DMAP (778 mg, 6.371 mmol) andpyridine (30.2 g, 31 mL, 382.3 mmol) under Ar at 0° C. The mixture wasslowly brought to room temperature. After 21 h, the reaction was cooledto 0° C., diethylamine (3.8 g, 4.2 mL, 63.7 mmol) was added dropwise,allowed to reach room temperature and stirred overnight. The mixture wasconcentrated, suspended in a mixture of 30% ethyl acetate in hexanes,passed trough a pad of SiO₂, and concentrated to yield a yellow oil(23.12 g, 95% yield). R_(f)=0.49 (30% ethyl acetate in hexanes); IR(neat) 1787, 1723, 1449, 1267, 1111 cm⁻¹; Formula C₂₂H₂₀O₆; MW 380.3906;¹H NMR (500 MHz, CDCl₃) δ 8.03-7.98 (m, 4H), 7.62 (td, J=7.4, 1.3 Hz,1H), 7.55 (td, J=7.4, 1.4 Hz, 1H), 7.47 (td, J=7.9, 7.5, 1.4 Hz, 2H),7.41-7.37 (m, 2H), 5.95 (dd, J=17.5, 10.7 Hz, 1H), 5.55 (d, J=7.5 Hz,1H), 5.44-5.30 (m, 2H), 4.76-4.71 (m, 2H), 4.59-4.54 (m, 1H), 1.56 (s,3H); ¹³C NMR (125 MHz, CDCl₃) δ 175.1, 166.0, 165.5, 134.0, 133.5,132.6, 130.0, 129.9, 128.8, 128.6, 118.5, 77.2, 63.2, 51.0, 21.6; ¹H NMR(500 MHz, C₆D₆) δ 8.04 (dd, J=8.3, 1.3 Hz, 2H), 7.96 (dd, J=8.3, 1.3 Hz,2H), 7.17-7.12 (m, 1H), 7.11-7.06 (m, 1H), 7.07-7.01 (m, 2H), 6.99 (ddd,J=8.2, 6.8, 1.2 Hz, 2H), 5.68 (dd, J=17.5, 10.7 Hz, 1H), 5.40 (d, J=7.6Hz, 1H), 5.23 (d, J=17.5 Hz, 1H), 5.03 (d, J=10.7 Hz, 1H), 4.46 (dd,J=12.1, 3.5 Hz, 1H), 4.38 (ddd, J=7.7, 5.8, 3.4 Hz, 1H), 4.30 (dd,J=12.1, 5.8 Hz, 1H), 1.38 (s, 3H); ¹³C NMR (125 MHz, C₆D₆) δ 174.4,165.8, 165.3, 133.7, 133.3, 133.2, 130.1, 130.0, 128.8, 128.6, 117.9,77.4, 77.4, 77.1, 63.6, 50.9, 21.5; MS (ESI) m/z 403.1 (M+Na⁺, 50),398.2 (M+NH₄ ⁺, 100), 381.1 (M+H⁺, 46); HRMS calcd for [M+H⁺]: 381.1338,found: 381.1317; calcd for [M+NH₄ ⁺]: 398.1604, found: 398.1580; calcdfor [M+Na⁺]: 403.1158, found: 403.1137; [α]_(D) +90 (c 2.0, CH₂Cl₂).

Example 2.6—Intermediate Compound

((2R,3S,4R)-3-(benzoyloxy)-5-hydroxy-4-methyl-4-vinyltetrahydrofuran-2-yl)methylbenzoate

LiAlH(OtBu)₃ (45 mL, 45 mmol, 1M in THF) was added dropwise at 0° C. tosolution of lactone (13.16 g, 34.60 mmol) in THF (115 mL) under Ar. Themixture was slowly brought to room temperature. After stirring for 72hours, Na₂SO₄.10H₂O (16.7 g, 51.90 mmol) was added at room temperatureand stirred vigorously for 1 h. The mixture was concentrated, suspendedin ethyl acetate and filtered through a pad of celite-SiO₂, washed withethyl acetate and concentrated to yield a clear yellow oil (11.08 g,83%, mixture of anomers in a 1.4:1 ratio of anomers). R_(f)=0.3 (×2, 20%ethyl acetate in hexanes); IR (neat) 3457, 1723, 1449, 1272, 1116 cm⁻¹;Formula C₂₂H₂₂O₆; MW 382.4065; For mixture of both anomers: ¹H NMR (500MHz, CDCl₃) δ 8.08-7.98 (m, 9H), 7.60-7.35 (m, 17H), 6.29 (dd, J=17.8,11.0 Hz, 1H), 6.10 (dd, J=17.6, 11.0 Hz, 1.4H), 5.57 (d, J=6.8 Hz,1.4H), 5.36-5.15 (m, 8H), 4.77-4.57 (m, 7H), 4.42 (td, J=6.5, 4.2 Hz,1.4H), 3.20 (dd, J=3.3, 1.7 Hz, 1H), 2.99 (dd, J=5.3, 1.4 Hz, 1H), 1.35(x 2s, 7H); ¹³C NMR (125 MHz, CDCl₃) δ 166.6, 166.1, 138.1, 135.3,133.5, 133.2, 133.1, 129.9, 128.6, 128.6, 128.5, 128.5, 117.8, 116.3,104.4, 103.4, 81.1, 80.8, 79.9, 79.6, 66.4, 65.0, 52.6, 51.8, 20.8,16.9; MS (ESI) m/z 405.1 (M+Na⁺, 43), 400.2 (M+NH₄ ⁺, 24), 365.1 (100);HRMS calcd for [M+NH₄ ⁺]: 400.1760, found: 400.1755; calcd for [M+Na⁺]:405.1314, found: 405.1311.

Example 2.7—Intermediate Compound

(3R,4S,5R)-5-((benzyloxy)methyl)-3-methyl-3-vinyltetrahydrofuran-2,4-diyldibenzoate

Benzoyl chloride (7.7 g, 6.4 mL, 55.0 mmol) was added slowly to amixture of lactols (16.18 g, 42.31 mmol), DMAP (517 mg, 4.23 mmol) andpyridine (10.0 g, 10.3 mL, 126.93 mmol) under Ar at 0° C. The mixturewas slowly brought to room temperature. After 21 h, 0.2 equiv of benzoylchloride (1.19 g, 0.98 mL, 8.46 mmol) was added. After 42 h, thereaction was cooled to 0° C., diethylamine (1.3 g, 1.4 mL, 21.16 mmol)was added dropwise (yellow precipitate formed). The mixture wasconcentrated, suspended in a mixture 20% ethyl acetate in hexanes,passed through a pad of SiO₂, concentrated to yield a clear yellow oil(18.06 g, 88% yield, 3:1 mixture of anomers) and another fractioncontaining 3 diastereomers (1.73 g in a 16:27:57 ratio, the first 2coming from 3,4-anti aldol adduct and the last from 3,4-syn aldoladduct). R_(f)=0.4 (30% ethyl acetate in hexanes); IR (neat) 3065, 2968,1728, 1599, 1449, 1272 cm⁻¹; Formula C₂₉H₂₆O₇; MW 486.5125; For majoranomer: ¹H NMR (500 MHz, CDCl₃) δ 8.14 (dd, J=8.4, 1.4 Hz, 1H), 8.05(ddd, J=13.9, 8.3, 1.4 Hz, 4H), 7.93-7.90 (m, 2H), 7.59 (ddt, J=7.7,6.1, 1.6 Hz, 2H), 7.47-7.41 (m, 4H), 7.20 (t, J=7.8 Hz, 2H), 6.43 (s,1H), 6.18 (dd, J=17.5, 11.2 Hz, 1H), 5.81 (d, J=7.6 Hz, 1H), 5.45 (s,1H), 5.42 (d, J=7.3 Hz, 1H), 4.70 (dd, J=11.5, 4.1 Hz, 1H), 4.60 (ddd,J=7.5, 5.6, 4.1 Hz, 1H), 4.54 (dd, J=11.6, 5.6 Hz, 1H), 1.42 (s, 3H);¹³C NMR (125 MHz, CDCl₃) δ 166.2, 165.8, 165.2, 136.5, 133.6, 133.5,133.0, 130.6, 129.9, 129.9, 129.7, 129.3, 128.9, 128.6, 128.6, 128.2,117.2, 102.1, 79.6, 78.5, 65.1, 52.1, 17.5; MS (ESI) m/z 509.2 (M+Na⁺,84), 504.2 (M+NH₄′, 100), 365.1 (61); HRMS calcd for [M+NH₄+]: 504.2022,found: 504.2021; calcd for [M+Na⁺]: 509.1576, found: 509.1576.

Example 2.8—Intermediate Compound

(3S,4S,5R)-5-((benzoyloxy)methyl)-3-formyl-3-methyltetrahydrofuran-2,4-diyldibenzoate

O₃ was flowed into a mixture of benzoylated lactols (18.06 g, 37.12mmol) in CH₂Cl₂ (150 mL) and pyridine (8.8 g, 9.0 mL, 111.36 mmol) at−78° C. After 5.5 h, the excess O₃ was removed under vacuum, a balloonwith N₂ was attached, Et₃N (3.7 g, 5.2 mL, 37.12 mmol) was added, andallowed to reach room temperature. The mixture was concentrated, dilutedwith ethyl acetate, washed (×1) with citric acid [0.1 M], (×1) NaHCO₃saturated solution, and dried over MgSO₄ to yield the title compound asa mixture of anomers in a 4:1 ratio (clear oil, 16.9 g, 93%). R_(f)=0.15(20% ethyl acetate in hexanes); IR (neat) 1728, 1599, 1449, 1267 cm⁻¹;Formula C₂₈H₂₄O₈; MW 488.4854; ¹H NMR (500 MHz, CDCl₃) δ 10.06 (s, 1H),9.97 (s, 4H), 8.08-7.23 (m, aromatics), 6.70 (s, 4H), 6.54 (s, 1H), 5.88(d, J=6.7 Hz, 4H), 5.48 (d, J=3.7 Hz, 1H), 4.87-4.56 (m, 15H), 1.50 (s,3H), 1.48 (s, 12H); ¹³C NMR (125 MHz, CDCl₃) δ 198.2, 197.5, 166.0,165.8, 165.5, 165.4, 164.6, 164.6, 133.8, 133.8, 133.6, 133.6, 133.1,132.9, 132.8, 129.7, 129.7, 129.7, 129.7, 129.6, 129.6, 129.6, 129.5,129.5, 129.4, 129.4, 129.2, 129.0, 128.7, 128.6, 128.6, 128.5, 128.5,128.4, 128.4, 128.3, 128.3, 128.2, 128.1, 128.0, 101.7, 98.5, 84.7,80.8, 80.1, 79.2, 64.7, 63.7, 60.3, 58.1, 17.9, 13.3; MS (ESI) m/z 511.1(M+Na⁺, 100); HRMS calcd for [M+NH₄ ⁺]: 506.1815, found: 506.1800; calcdfor [M+Na⁺]: 511.1369, found: 511.1362.

Example 2.9—Intermediate Compound

(3R,4S,5R)-5-((benzoyloxy)methyl)-3-(hydroxymethyl)-3-methyltetrahydrofuran-2,4-diyldibenzoate

NaBH₄ (1.34 g, 34.64 mmol) was slowly added in portions to a mixture ofaldehydes (16.92 g, 34.64 mmol) in THF/MeOH (1:2) (345 mL) under Ar at0° C. After 2 h, the reaction was quenched at 0° C. by addition of 20 mLof distilled water and stirred at room temperature for 40 minutes. Themixture was concentrated, suspended in ethyl acetate, and washed withdistilled water. The aqueous phase was back extracted with ethyl acetate(×3), the organics were mixed and dried over MgSO₄, concentrated andpurified by silica gel column chromatography (30% ethyl acetate inhexanes) to yield the title compound as a white solid (12.26 g, 72%,mixture of anomers 1.6:1). R_(f)=0.2 (30% ethyl acetate in hexanes); IR(neat) 3483, 3070, 2947, 1723, 1599, 1449, 1272 cm⁻¹; Formula C₂₈H₂₆O₈;MW 490.5012; For major anomer: ¹H NMR (500 MHz, CDCl₃) δ 8.08 (dd,J=8.0, 1.4 Hz, 2H), 7.99-7.94 (m, 4H), 7.61-7.54 (m, 2H), 7.50 (t, J=7.6Hz, 1H), 7.45 (t, J=7.7 Hz, 2H), 7.38 (t, J=7.7 Hz, 2H), 7.28 (t, J=8.0Hz, 2H), 6.59 (s, 1H), 4.62-4.51 (m, 5H), 4.42 (dd, J=8.0, 5.5 Hz, 1H),4.37 (dt, J=8.1, 4.2 Hz, 1H), 1.35 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ167.0, 166.8, 165.4, 133.5, 133.4, 133.3, 129.9, 129.9, 129.8, 128.7,128.7, 128.5, 100.7, 82.4, 77.9, 65.6, 65.2, 49.9, 16.4; MS (ESI) m/z508.2 (M+NH₄ ⁺, 98), 369.1 (100); HRMS calcd for [M+NH₄ ⁺]: 508.1971,found: 508.1970; calcd for [M+Na⁺]: 513.1525, found: 513.1518.

For mixture of anomers: ¹H NMR (500 MHz, CDCl₃) δ 8.11-7.32 (m, 19H),7.28 (t, J=7.9 Hz, 1H), 7.22 (t, J=7.8 Hz, 1H), 6.60 (d, J=4.8 Hz, 1H),5.79 (d, J=6.4 Hz, 0.6H), 4.72-4.31 (m, 8H), 4.02 (d, J=11.3 Hz, 0.6H),3.95-3.91 (m, 1H), 1.35 (5H); ¹³C NMR (125 MHz, CDCl₃) δ 167.0, 166.8,166.3, 165.9, 165.5, 165.4, 133.8, 133.6, 133.5, 133.4, 133.3, 133.1,129.9, 129.9, 129.8, 129.8, 129.8, 129.8, 129.7, 129.7, 129.6, 128.8,128.7, 128.7, 128.6, 128.5, 128.4, 128.3, 101.4, 100.7, 82.4, 80.8,79.1, 77.6, 65.6, 65.6, 65.2, 64.7, 51.4, 49.8, 16.4, 16.2.

Example 2.10—Intermediate Compound

(3R,4S,5R)-3,5-bis((benzoyloxy)methyl)-3-methyltetrahydrofuran-2,4-diyldibenzoate

Benzoyl chloride (6.6 g, 5.4 mL, 46.89 mmol) was added slowly to amixture of alcohols (11.5 g, 23.44 mmol), DMAP (286 mg, 2.34 mmol) andpyridine (7.4 g, 7.6 mL, 93.78 mmol) under Ar at 0° C. The mixture wasslowly brought to room temperature. After 21 h, the reaction was cooledto 0° C., diethylamine (1.41 g, 1.57 mL, 23.44 mmol) was added dropwiseand stirred for 4 h at rt. The mixture was concentrated, suspended in amixture of 20% ethyl acetate in hexane, passed through a pad of SiO₂,and concentrated to yield a white foam (12.73 g, 91%, mixture of anomerswith an 8:1 ratio). R_(f)=0.38 (30% ethyl acetate in hexanes); IR (neat)1728, 1449, 1261, 1106 cm⁻¹; Formula C₃₅H₃₀O₉; MW 594.6073; For majoranomer: ¹H NMR (500 MHz, CDCl₃) δ 8.11-8.00 (m, 6H), 7.96-7.88 (m, 2H),7.63-7.52 (m, 3H), 7.44 (td, J=7.8, 4.5 Hz, 7H), 7.23 (t, J=7.7 Hz, 2H),6.72 (s, 1H), 5.90 (d, J=6.6 Hz, 1H), 4.75-4.52 (m, 5H), 1.47 (s, 3H);¹³C NMR (125 MHz, CDCl₃) δ 166.4, 166.2, 165.8, 165.3, 133.9, 133.7,133.5, 133.1, 130.0, 130.0, 130.0, 129.8, 129.8, 129.8, 128.8, 128.7,128.3, 101.0, 80.7, 78.6, 65.7, 65.5, 50.3, 16.7; MS (ESI) m/z 1211.4(100), 617.2 (M+Na⁺, 75), 612.2 (M+NH₄ ⁺, 63), 473.2 (66); HRMS calcdfor [M+NH₄ ⁺]: 612.2234, found: 612.2234; calcd for [M+Na⁺]: 617.1788,found: 617.1789.

Example 2.11—Intermediate Compound

((2R,3S,4R,5R)-3-(benzoyloxy)-5-(2,6-dichloro-9H-purin-9-yl)-4-methyltetrahydrofuran-2,4-diyl)bis(methylene)dibenzoate

DBU (3.67 mL, 3.66 g, 24.06 mmol) was added to a mixture of benzoylatedsugar (4.77 g, 8.02 mmol), and 2,6-dichloropurine (1.67 g, 8.82 mmol) indry acetonitrile (32 mL), under N₂ at −10° C. The mixture was stirredand then TMSOTf (5.9 mL, 7.26 g, 32.08 mmol) was added dropwise over 2minutes. After 3 h, the reaction was quenched by addition of saturatedsolution of NaHCO₃ (5 mL) at −10° C., suspended in CH₂Cl₂, and washed ×1with a saturated solution of NaHCO₃. The aqueous phase was extractedwith CH₂Cl₂ (×3), the organics were washed ×2 with citric acid [0.1M],dried over MgSO₄, and concentrated to produce beige foam. ¹H-NMR of thecrude showed a 8:1 ratio of β:α anomers. The crude was fractionated bysilica gel column chromatography (20% ethyl acetate in hexanes) toprovide a white solid as a 10:1 mixture of β:α anomers (3.96 g, 74%).R_(f)=0.32 (30% ethyl acetate in hexanes); IR (neat) v_(max) 3065, 2968,1723, 1589, 1551, 1267 cm⁻¹; Formula C₃₃H₂₆Cl₂N₄O₇; MW 661.4881; ¹H NMR(500 MHz, CDCl₃) δ 8.44 (s, 1H), 8.05 (ddd, J=8.0, 6.4, 1.4 Hz, 4H),7.76-7.71 (m, 2H), 7.65-7.51 (m, 3H), 7.45 (dt, J=10.7, 7.8 Hz, 4H),7.37 (t, J=7.8 Hz, 2H), 6.62 (s, 1H), 5.70 (d, J=5.9 Hz, 1H), 4.93-4.84(m, 3H), 4.63 (q, J=4.8 Hz, 1H), 4.52 (d, J=11.6 Hz, 1H), 1.23 (s, 3H);¹³C NMR (125 MHz, CDCl₃) δ 166.3, 165.8, 165.7, 153.4, 152.5, 152.2,144.1, 134.2, 133.6, 133.6, 130.9, 130.0, 129.8, 129.5, 129.3, 129.1,128.9, 128.8, 128.6, 128.4, 89.1, 81.1, 78.2, 65.7, 63.5, 49.3, 17.6; MS(ESI) m/z 683.1 (M+Na⁺, 100), 360.3 (86), 226.9 (24); HRMS calcd forC₃₃H₂₆Cl₂N₄NaO₇ [M+Na⁺]:683.1091 found: 683.1066.

Example 2.12—Intermediate Compound

((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-hydroxy-4-methyltetrahydrofuran-2,4-diyl)dimethanol

NH₃ in MeOH [7N] (56 mL, 392.0 mmol) was added to Bz-nucleoside (3.88 g,5.86 mmol) under N₂ at room temperature. After 3 days, the reactionmixture was concentrated to produce brown oil. The crude wasfractionated by silica gel column chromatography (MeOH in CH₂Cl₂ 5%-20%)to yield the title compound as a mixture of anomers β:α (12:1) (60%,beige solid). R_(f)=0.17 (10% MeOH in CH₂Cl₂); IR (neat) v_(max) 3339,1653, 1594, 1309, 1036 cm⁻¹; Formula C₁₂H₁₆ClN₅O₄; MW 329.7395; ¹H NMR(500 MHz, CD₃OD) δ 8.44 (s, 1H), 6.26 (s, 1H), 4.81 (s, 1H), 4.36 (d,J=8.7 Hz, 1H), 4.07 (ddd, J=8.8, 3.7, 2.3 Hz, 1H), 3.97 (dd, J=12.4, 2.4Hz, 1H), 3.90-3.83 (m, 3H), 3.77 (d, J=11.2 Hz, 1H), 0.70 (s, 3H); ¹³CNMR (125 MHz, CD₃OD) δ 158.1, 155.2, 151.5, 141.9, 119.0, 90.6, 85.6,76.3, 65.4, 61.7, 51.6, 17.3; MS (ESI) m/z 352.0 (M+Na⁺, 100), 187.0;HRMS calcd for [M+H⁺]: 330.0969, found: 330.0959; calcd for [M+Na⁺]:352.0789, found: 352.0783; [α]_(D) −10 (c 1.0, MeOH).

Example 2.13—Intermediate Compound

((2R,3S,4R,5R)-5-(2-chloro-6-methoxy-9H-purin-9-yl)-3-hydroxy-4-methyltetrahydrofuran-2,4-diyl)dimethanol

A small amount of the title compound was also isolated and characterizedafter purification of the above crude mixture. The pure β-anomer wassuccessfully crystallized in ethyl acetate (EtOAc) and proof ofstructure was obtained by X-ray analysis. R_(f)=0.35 (DCM/MeOH, 90:10);[α]²⁵ _(D) +14 (c 0.8, MeOH); Formula: C₁₃H₁₇ClN₄O₅; MW: 344.75 g/mol;IR (neat) v_(max) 3335, 2933, 2880, 1598, 1471 cm⁻¹; ¹H NMR (500 MHz,CD₃OD) δ 8.76 (s, 1H), 6.40 (s, 1H), 4.37 (d, J=8.7 Hz, 1H), 4.20 (s,3H), 4.13-4.06 (m, 1H), 3.99 (dd, J=12.4, 2.3 Hz, 1H), 3.93-3.85 (m,2H), 3.79 (d, J=11.3 Hz, 1H), 0.70 (s, 3H) ppm (Labile protons were notobserved due to exchange with deuterated solvent); ¹³C NMR (125 MHz,CD₃OD) δ 161.4, 153.1, 152.6, 143.0, 119.8, 89.5, 84.6, 74.9, 64.1,60.4, 54.5, 50.6, 16.1 ppm; HRMS calcd for: C₁₃H₁₇ClN₄NaO₅[M+Na]⁺:367.0780; found 367.0781 (0.46 ppm).

Example 2.14—Intermediate Compound

((2R,3S,4R,5R)-5-(2-amino-6-chloro-9H-purin-9-yl)-3-(benzoyloxy)-4-methyltetrahydrofuran-2,4-diyl)bis(methylene)dibenzoate

DBU (151 μL, 151 mg, 0.989 mmol) was added to a mixture of benzoylatedsugar (202 mg, 0.330 mmol), and 2-amino-6-chloropurine (61.5 mg, 0.363mmol) in dry acetonitrile (3.8 mL), under N₂ at 0° C. The mixture wasstirred and then TMSOTf (243 μL, 299 mg, 1.32 mmol) was added dropwiseover 5 minutes. After 15 minutes, the reaction was heated at 70° C. for1 h, then diluted with CH₂Cl₂, and washed ×1 with a saturated solutionof NaHCO₃. The aqueous phase was extracted with CH₂Cl₂ (×3), theorganics dried over MgSO₄, and concentrated. DBU was removed by passingthe crude through a pad of SiO₂ and washing with 2% MeOH in CH₂Cl₂ toproduce an inseparable mixture of 4 compounds (83%, 177 mg, 67 (N-9 β):13 (N-9 α) ratio and two other isomers). R_(f)=0.4 (50% ethyl acetate inhexanes); Formula C₃₃H₂₈ClN₅O₇; MW 642.0577;

For major compound:

¹H NMR (500 MHz, CDCl₃) δ 8.06-7.97 (m, 5H), 7.84-7.77 (m, 2H),7.61-7.51 (m, 4H), 7.39 (ddd, J=13.8, 8.6, 6.9 Hz, 5H), 6.40 (s, 1H),5.81 (d, J=5.9 Hz, 1H), 4.98 (dd, J=12.0, 4.0 Hz, 1H), 4.83-4.74 (m,2H), 4.61 (td, J=5.8, 3.9 Hz, 1H), 4.52 (d, J=11.5 Hz, 1H), 1.20 (s,3H); ¹³C NMR (125 MHz, CDCl₃) δ 166.4, 166.0, 165.7, 159.1, 153.1,151.7, 140.5, 134.0, 133.6, 133.5, 129.9, 129.8, 129.4, 128.8, 128.7,128.6, 125.4, 88.9, 80.7, 78.7, 66.0, 63.7, 49.2, 17.5; MS (ESI) m/z664.2 (M+Na⁺, 48), 360.3 (100), 227.0 (29); HRMS calcd for [M+H⁺]:642.1756, found: 642.1746; calcd for [M+Na⁺]: 664.1575, found: 664.1568.

Example 2.15—Intermediate Compound

((2R,3S,4R,5R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3-hydroxy-4-methyltetrahydrofuran-2,4-diyl)dimethanol

NaOMe in MeOH (25% wt) (73.5 μL, 0.321 mmol) was added to Bz-nucleoside(68.8 mg, 0.107 mmol) under Ar at room temperature. After 64 h, thereaction mixture was neutralized with HCl (2N), diluted in MeOH andconcentrated. The crude was purified by reverse phase columnchromatography (MeOH in H₂O 0%-100%) to yield the title compound as amixture of isomers (82 (N-9 β): 11 (N-9 α)) (68%, 23.7 mg). R_(f)=0.05(10% MeOH in CH₂Cl₂); Formula C₁₃H₁₉N₅O₅; MW 325.3205; ¹H NMR (500 MHz,CD₃OD) δ 8.21 (s, 1H), 6.22 (s, 1H), 4.38-4.31 (m, 1H), 4.10-3.94 (m,5H), 3.89-3.81 (m, 2H), 3.75 (dd, J=11.3, 1.5 Hz, 1H), 0.71 (s, 3H); ¹³CNMR (125 MHz, CD₃OD) δ 162.7, 161.7, 154.4, 139.9, 115.2, 90.3, 85.4,76.4, 65.6, 61.7, 54.2, 51.5, 17.3; MS (ESI) m/z 348.1 (M+Na⁺, 55),326.1 (40), 226.9 (26), 166.0 (100); HRMS calcd for [M+H⁺]: 326.1464,found: 326.1467; calcd for [M+Na⁺]: 348.1284, found: 348.1287.

Example 2.16—Intermediate Compound

((4aR,5R,7R,7aS)-5-(6-amino-2-chloro-9H-purin-9-yl)-2,2,4a-trimethyltetrahydro-4H-furo[3,4-d][1,3]dioxin-7-yl)methanol

2,2-dimethoxy propane (6.0 g, 7.0 mL, 57.15 mmol) was added to a mixtureof nucleoside (1.05 g, 3.18 mmol), CSA (737 mg, 3.18 mmol), molecularsieves 3 Å (3 g), and dry acetone (32 mL). After 16.5 h, the reactionmixture was concentrated, diluted in CH₂Cl₂, and fractionated by silicaget column chromatography (10% methanol in dichloromethane) to yield twofractions: A) 365 mg (mixture 2:1, β:α anomers) and B) 374 mg (whitesolid, only β-anomer), 63% overall yield. R_(f)=0.36 (MeOH in CH₂Cl₂10%); IR (neat) v_(max) 3322, 3172, 2984, 2936, 1648, 1594, 1304, 1084cm⁻¹; Formula C₁₅H₂₀ClN₅O₄; MW 369.8034; ¹H NMR (500 MHz, CD₃OD) δ 8.31(s, 1H), 6.31 (s, 1H), 4.17-4.07 (m, 3H), 3.89-3.77 (m, 2H), 3.62 (d,J=12.2 Hz, 1H), 1.44 (d, J=3.8 Hz, 6H), 0.80 (s, 3H); ¹³C NMR (125 MHz,CD₃OD) δ 158.0, 155.5, 151.4, 141.0, 118.9, 100.2, 90.6, 86.8, 79.4,65.4, 63.1, 46.9, 27.2, 21.5, 16.8; MS (ESI) m/z 392.1 (M+Na⁺, 100),370.1 (16) 226.9; HRMS calcd for [M+H⁺]: 370.1282, found: 370.1278;calcd for [M+Na⁺]: 392.1102, found: 392.1102.

Example 2.17—Intermediate Compound

((4aR,5R,7R,7aS)-5-(6-amino-2-chloro-9H-purin-9-yl)-2,2,4a-trimethyltetrahydro-4H-furo[3,4-d][1,3]dioxin-7-yl)methyldiethyl phosphate

t-BuMgCl (2.5 mL, 2.47 mmol, 1 in THF) was added dropwise to a solutionof nucleoside (365 mg, 0.987 mmol) in anhydrous THF (5.8 mL) under N₂ atroom temperature. After addition, a clear yellow solution was formed.After 30 minutes, diethylchlorophosphate (426 mg, 357 μL, 2.47 mmol) wasadded dropwise. After 17.5 h, the reaction was quenched with MeOH (5mL), concentrated, and fractionated by silica gel column chromatography(100% ethyl acetate, then 5%-10%-20% methanol in dichloromethane) toyield the product as a white solid (469 mg, 94% yield). R_(f)=0.6 (10%MeOH in CH₂Cl₂); IR (neat) v_(max) 3323, 3182, 2987, 1650, 1595, 1302,1254, 1031 cm⁻¹; Formula C₁₉H₂₉ClN₅O₇P; MW 505.8896; ¹H NMR (500 MHz,CDCl₃) δ 8.16 (s, 1H), 6.46 (dt, J=26.5, 12.1 Hz, 2H), 6.32 (s, 1H),4.36-4.28 (m, 2H), 4.26 (dt, J=4.4, 2.0 Hz, 1H), 4.20-4.12 (m, 5H), 4.03(d, J=2.4 Hz, 1H), 3.59 (d, J=12.2 Hz, 1H), 1.42 (d, J=25.3 Hz, 6H),1.39-1.32 (m, 6H), 0.79 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 156.4,154.3, 150.3, 139.4, 118.5, 99.4, 89.6, 82.7, 82.7, 78.1, 66.6, 66.5,64.7, 64.3, 64.3, 46.4, 26.5, 21.6, 16.8, 16.3, 16.2; MS (ESI) m/z 528.1(M+Na⁺, 100), 506.1 (27); HRMS calcd for [M+H⁺]: 506.1571, found:506.1565; calcd for [M+Na⁺]: 528.1391, found: 528.1386; [α]_(D) +35 (c1.0, MeOH).

Example 2.18—Cardioprotective Compound-LCB2122

((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-hydroxy-4-(hydroxymethyl)-4-methyltetrahydrofuran-2-yl)methyldiethyl phosphate (LCB2122)

A mixture of TFA/H₂O (8:2) (7.1 mL) was added to a solution of theprodrug (360 mg, 0.711 mmol) in THF (7.1 mL) at room temperature andopen atmosphere. After 1 h, the solvent was removed under vacuum,concentrated ×3 from MeOH and purified by silica gel columnchromatography (MeOH in CH₂Cl₂ 5-20%) to yield the title compound aswhite solid (294 mg, 88% yield). R_(f)=0.2 (10% MeOH in CH₂Cl₂); IR(neat) v_(max) 3327, 3156, 2984, 1648, 1596, 1313, 1243, 1029 cm⁻¹;Formula C₁₆H₂₅ClN₅O₇P; MW 465.8258; ¹H NMR (500 MHz, CD₃OD) δ 8.18 (s,1H), 6.27 (s, 1H), 4.56-4.47 (m, 1H), 4.45-4.33 (m, 2H), 4.30-4.23 (m,1H), 4.17-4.03 (m, 4H), 3.88 (d, J=11.2 Hz, 1H), 3.74 (d, J=11.2 Hz,1H), 1.29 (qd, J=7.0, 1.0 Hz, 6H), 0.72 (s, 3H); ¹³C NMR (125 MHz,CD₃OD) δ 158.2, 155.3, 151.4, 141.7, 119.3, 90.9, 83.7, 77.0, 68.4,65.6, 65.5, 65.5, 65.3, 51.5, 17.1, 16.4, 16.3, 16.3; MS (ESI) m/z 488.1(M+Na⁺, 100), 360.3 (15); HRMS calcd for [M+H⁺]: 466.1258, found:466.1247; calcd for [M+Na⁺]: 488.1078, found: 488.1072.

Example 2.19—Cardioprotective Compound-LCB2195

((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3-hydroxy-4-(hydroxymethyl)-4-methyltetrahydrofuran-2-yl)methyldiethyl phosphate (LCB2195)

To a solution of LCB2122 (22.9 mg, 0.0492 mmol) in MeOH (0.5 mL, 0.1 M)at room temperature was added palladium on carbon (10 wt. %) (22.9 mg)and the N_(2(g)) was removed from the reaction and replaced by apositive pressure of H₂. After 6 hours at room temperature, the mixturewas filtered over celite with MeOH/EtOAc (50:50) and concentrated invacuo. Crude product was purified by reverse phase (C18) flashchromatography (H₂O/MeCN, 60:40 to give the final product as a whitesolid (15.2 mg, 72% yield). R_(f)=0.2 (DCM/MeOH, 95:5); [α]²⁵ _(D) −2 (c0.8, MeOH); Formula: C₁₆H₂₆N₅O₇P; MW: 431.39 g/mol; IR (neat) v_(max)3330, 3195, 2981, 1647, 1600 cm⁻¹; ¹H NMR (500 MHz, CD₃OD) δ 8.27 (s,1H), 8.23 (s, 1H), 6.38 (s, 1H), 4.51-4.36 (m, 3H), 4.32-4.26 (m, 1H),4.20-4.09 (m, 4H), 3.90 (d, J=11.2 Hz, 1H), 3.77 (d, J=11.2 Hz, 1H),1.32 (tdd, J=7.1, 2.0, 1.0 Hz, 6H), 0.71 (s, 3H) ppm (Labile protonswere not observed due to exchange with deuterated solvent); ¹³C NMR (125MHz, CD₃OD) δ 156.0, 152.5, 149.0, 139.9, 118.8, 89.1, 82.1 (d, J=7.5Hz), 75.4, 66.8 (d, J=5.8 Hz), 64.23 (d, J=5.7 Hz), 64.18 (d, J=5.8 Hz),63.9, 50.1, 15.7, 15.0 (d, J=5.1 Hz), 14.9 (d, J=5.0 Hz) ppm; HRMS calcdfor: C₁₆H₂₇N₅O₇P [M+H]⁺: 432.1643; found 432.1642 (−0.13 ppm).

Example 2.20—Intermediate Compound

((4aR,5R,7R,7aS)-5-(2-chloro-6-methoxy-9H-purin-9-yl)-2,2,4a-trimethyltetrahydro-4H-furo[3,4-d][1,3]dioxin-7-yl)methanol

2,2-dimethoxy propane (1.60 mL, 18.0 equiv., 13.1 mmol) was added to amixture of nucleoside (250 mg, 725 umol), CSA (168 mg, 725 umol),molecular sieves 3 Å (500 mg), and dry acetone (8 mL). After 16.5 h, thereaction mixture was concentrated, diluted in CH₂Cl₂, and purified bysilica gel column chromatography (5-10% methanol in dichloromethane) toyield the product 278 mg (99.6%) as a white/yellow powder. FormulaC₁₆H₂₁ClN₄O₅; MW 384.8170; ¹H NMR (500 MHz, CD₃OD) δ 8.53 (s, 1H), 6.41(s, 1H), 4.19 (d, J=0.7 Hz, 4H), 4.17 (d, J=2.2 Hz, 1H), 4.14-4.12 (m,1H), 3.87 (dd, J=12.2, 4.5 Hz, 1H), 3.81 (dd, J=12.1, 5.3 Hz, 1H), 3.65(d, J=12.3 Hz, 1H), 1.45 (s, 3H), 1.45 (s, 3H), 0.80 (s, 3H) (Labileprotons were not observed due to exchange with deuterated solvent).

Example 2.21—Intermediate Compound

((4aR,5R,7R,7aS)-5-(2-chloro-6-methoxy-9H-purin-9-yl)-2,2,4a-trimethyltetrahydro-4H-furo[3,4-d][1,3]dioxin-7-yl)methyldiethyl phosphate

t-BuMgCl (1.08 mL, 1.50 equiv., 1.08 mmol, 1M in THF) was added dropwiseto a solution of nucleoside (278 mg, 722 umol) in anhydrous THF (7.2 mL)under N₂ at room temperature. After addition, a clear yellow solutionwas formed. After 40 minutes, diethylchlorophosphate (157 μL, 1.50equiv., 1.08 mmol) was added dropwise. After 5 h, the reaction wasquenched with MeOH, concentrated, and purified by silica gel columnchromatography (5%-10% methanol in dichloromethane) to yield the productas a yellow oil (262 mg, 70% yield). Formula C₂₀H₃₀ClN₄O₈P; MW 520.9038;¹H NMR (500 MHz, CD₃OD) δ 8.52 (s, 1H), 6.50 (s, 1H), 4.42-4.35 (m, 2H),4.24 (d, J=2.0 Hz, 1H), 4.23-4.14 (m, 7H), 4.14-4.09 (m, 2H), 3.70 (d,J=12.4 Hz, 1H), 1.47 (s, 6H), 1.40-1.27 (m, 6H), 0.81 (s, 3H).

Example 2.22—Cardioprotective Compound-LCB2223

((2R,3S,4R,5R)-5-(2-chloro-6-methoxy-9H-purin-9-yl)-3-hydroxy-4-(hydroxymethyl)-4-methyltetrahydrofuran-2-yl)methyldiethyl phosphate (LCB2223)

A mixture of TFA/H₂O (8:2) (5.2 mL, 0.1M) was added to a solution of theprodrug (262 mg, 503 umol) in THF (5.2 mL, 0.1 M) at room temperatureand open atmosphere. After 3 h, MeOH was added and the solvent wasremoved under vacuum, concentrated ×3 from MeOH and purified by reversephase column chromatography (C18) (H₂O/MeCN, 60:40) to yield the titlecompound as white solid (128 mg, 53% yield). R_(f)=0.4 (10% MeOH inCH₂Cl₂); [α]²⁵ _(D) +13 (c 0.7, MeOH); Formula: C₁₇H₂₆ClN₄O₈P; MW:480.8388 g/mol; IR (neat) v_(max) 3333, 2983, 2359, 1597, 1472, 1387cm⁻¹; ¹H NMR (500 MHz, CD₃OD) δ 8.38 (s, 1H), 6.38 (s, 1H), 4.55-4.47(m, 1H), 4.44-4.36 (m, 2H), 4.32-4.26 (m, 1H), 4.19 (s, 3H), 4.15-4.09(m, 4H), 3.89 (d, J=11.2 Hz, 1H), 3.74 (d, J=11.2 Hz, 1H), 1.34-1.25 (m,6H), 0.70 (s, 3H) (Labile protons were not observed due to exchange withdeuterated solvent); ¹³C NMR (125 MHz, CD₃OD) δ 162.7, 154.3, 153.7,144.1, 121.4, 91.2, 83.83, 83.77, 76.9, 68.4 (d, J=5.8 Hz), 65.6 (t,J=5.3 Hz), 65.2, 55.7, 51.6, 17.1, 16.4 (d, J=4.7 Hz), 16.3 (d, J=4.7Hz); HRMS calcd for [M+Na⁺]: 503.1069, found: 503.1055 (−2.78 ppm).

Example 2.23—Intermediate Compound

((4aR,5R,7R,7aS)-5-(6-amino-2-chloro-9H-purin-9-yl)-2,2,4a-trimethyltetrahydro-4H-furo[3,4-d][1,3]dioxin-7-yl)methyldiisopropyl phosphate

To a solution of acetonide (34.7 mg, 0.0938 mmol) in THF (1 mL, 0.1 M)was added t-BuMgCl (141 μL, 0.141 mmol) dropwise at room temperature andthe mixture was stirred for 45 minutes. Diisopropylchlorophosphate (24.9μL, 0.141 mmol) was added slowly and the reaction was stirred for 4hours. The reaction was quenched by addition of MeOH and concentrated invacuo. Crude product was purified by reverse phase (C18) flashchromatography (H₂O/MeCN, 50:50) to give the product as a white solid(34.8 mg, 70% yield). R_(f)=0.55 (DCM/MeOH, 90:10); [α]²⁵ _(D) +25 (c2.36, MeOH); Formula: C₂₁H₃₃ClN₅O₇P; MW: 533.95 g/mol; IR (neat) v_(max)3324, 3182, 2983, 2937, 1650, 1595 cm⁻¹; ¹H NMR (500 MHz, CD₃OD) δ 8.32(s, 1H), 6.40 (s, 1H), 4.70 (hept, J=12.7, 6.3 Hz, 2H), 4.35 (dd, J=8.2,5.0 Hz, 2H), 4.32-4.27 (m, 1H), 4.23 (d, J=2.0 Hz, 1H), 4.12 (d, J=12.3Hz, 1H), 3.67 (d, J=12.4 Hz, 1H), 1.47 (s, 3H), 1.46 (s, 3H), 1.38 (t,J=6.4 Hz, 12H), 0.83 (s, 3H) ppm (Labile protons were not observed dueto exchange with deuterated solvent); ¹³C NMR (125 MHz, CD₃OD) δ 156.6,154.2, 150.0, 139.4, 117.5, 98.8, 89.0, 83.0 (d, J=7.0 Hz), 77.8, 73.5(d, J=2.8 Hz), 73.4 (d, J=2.5 Hz), 66.7 (d, J=5.8 Hz), 63.6, 44.9, 26.0,22.6, 22.54, 22.51, 22.50, 19.8, 15.5 ppm; HRMS calcd for: C₂₁H₃₄ClN₅O₇P[M+H]⁺: 534.1879; found 534.1868 (−1.97 ppm).

Example 2.24—Cardioprotective Compound-LCB2177

((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-hydroxy-4-(hydroxymethyl)-4-methyltetrahydrofuran-2-yl)methyldiisopropyl phosphate (LCB2177)

To a solution of nucleoside (34.8 mg, 0.0652 mmol) in THF (0.7 mL, 0.1M) was slowly added a mixture of TFA/H₂O (8:2) (0.7 mL, 0.1 M) at roomtemperature and open atmosphere. After 4 hours, MeOH was added and thereaction was concentrated in vacuo, then co-evaporated with MeOH (3×).Crude product was purified by reverse phase (C18) flash chromatography(H₂O/MeCN, 60:40) to give the final product as a white solid (23.9 mg,74% yield). R_(f)=0.3 (DCM/MeOH, 90:10); [α]²⁵ _(D) +7 (c 1.72, MeOH);Formula: C₁₈H₂₉ClN₅O₇P; MW: 493.88 g/mol; IR (neat) v_(max) 3344, 2984,2936, 2405, 1615 cm⁻¹; ¹H NMR (500 MHz, CD₃OD) δ 8.20 (s, 1H), 6.28 (s,1H), 4.71-4.58 (m, 2H), 4.50 (dt, J=11.6, 5.9 Hz, 1H), 4.43 (d, J=8.8Hz, 1H), 4.37 (ddd, J=11.3, 5.2, 2.1 Hz, 1H), 4.31-4.22 (m, 1H), 3.89(d, J=11.2 Hz, 1H), 3.76 (d, J=11.2 Hz, 1H), 1.33 (dd, J=6.3, 2.3 Hz,9H), 1.29 (d, J=6.2 Hz, 3H), 0.73 (s, 3H) ppm (Labile protons were notobserved due to exchange with deuterated solvent); ¹³C NMR (125 MHz,CD₃OD) δ 156.8, 153.9, 150.0, 140.4, 117.9, 89.5, 82.4 (d, J=8.0 Hz),75.5, 73.3 (d, J=2.9 Hz), 73.2 (d, J=2.8 Hz), 66.9 (d, J=5.9 Hz), 63.9,50.1, 22.4 (td, J=5.1, 3.5 Hz) (4C), 15.7 ppm; HRMS calcd for:C₁₈H₃₀ClN₅O₇P [M+H]⁺: 494.1566; found 494.1579 (2.70 ppm).

Example 2.25—Intermediate Compound

((4aR,5R,7R,7aS)-5-(6-amino-2-chloro-9H-purin-9-yl)-2,2,4a-trimethyltetrahydro-4H-furo[3,4-d][1,3]dioxin-7-yl)methyldimethyl phosphate

Following the same procedure as in example 2.23 usingDimethylchlorophosphate and purification by silica gel columnchromatography (5:95 MeOH:DCM), the product was obtained. R_(f)=0.57(DCM/MeOH, 95:5); [α]²⁵ _(D) +35 (c 0.9, CD₃OD); Formula: C₁₇H₂₅ClN₅O₇P;MW: 477.84 g/mol; IR (neat) v_(max) 3365, 3182, 2989, 2936, 1610 cm⁻¹;¹H NMR (500 MHz, (CD₃)₂SO) δ 8.36 (s, 1H), 7.87 (s, 2H), 6.35 (s, 1H),4.31 (dd, J=8.6, 6.1 Hz, 2H), 4.26 (d, J=1.4 Hz, 1H), 4.20-4.14 (m, 1H),3.86 (d, J=12.5 Hz, 1H), 3.72 (d, J=3.6 Hz, 3H), 3.70 (d, J=3.7 Hz, 3H),3.61 (d, J=12.4 Hz, 1H), 1.43 (s, 3H), 1.39 (s, 3H), 0.75 (s, 3H); ¹³CNMR (125 MHz, CD₃OD) δ 158.1, 155.6, 151.5, 140.9, 118.9, 100.2, 90.3,84.4 (d, J=6.2 Hz), 79.1, 68.7 (d, J=5.8 Hz), 64.9, 55.5 (d, J=6.2 Hz),55.4 (d, J=6.2 Hz), 46.1, 27.5, 21.1, 16.8 ppm; HRMS calcd for:C₁₇H₂₆ClN₅O₇P [M+H]⁺: 478.1253; found 478.1249 (−0.91 ppm).

Example 2.26—Cardioprotective Compound-LCB2234

((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-hydroxy-4-(hydroxymethyl)-4-methyltetrahydrofuran-2-yl)methyldimethyl phosphate (LCB2234)

Following the same procedure as in example 2.24 and purification byreverse phase column chromatography (H₂O:MeCN), the product wasobtained. R_(f)=0.38 (DCM/MeOH, 90:10); [α]²⁵ _(D) +4.1 (c 0.8, CD₃OD);Formula: C₁₄H₂₁ClN₅O₇P; MW: 437.77 g/mol; IR (neat) v_(max) 3333, 3210,2952, 1616 cm⁻¹; ¹H NMR (500 MHz, CD₃OD) δ 8.18 (s, 1H), 6.28 (s, 1H),4.57-4.51 (m, 1H), 4.43-4.37 (m, 2H), 4.29-4.24 (m, 1H), 3.88 (d, J=11.2Hz, 1H), 3.78 (d, J=6.6 Hz, 3H), 3.77 (d, J=6.6 Hz, 3H), 3.74 (d, J=11.2Hz, 1H), 0.72 (s, 3H) ppm (Labile protons were not observed due toexchange with deuterated solvent); ¹³C NMR (125 MHz, CD₃OD) δ 158.2,155.4, 151.4, 141.7, 119.3, 90.9, 83.6 (d, J=7.3 Hz), 77.0, 68.7 (d,J=5.7 Hz), 65.3, 55.31 (d, J=6.0 Hz), 55.25 (d, J=6.1 Hz), 51.6, 17.1ppm; HRMS calcd for: C₁₄H₂₂ClN₅O₇P [M+H]⁺: 438.0940; found 438.0946(1.42 ppm).

Example 2.27—Intermediate Compound

(3R,4S,5R)-5-((benzoyloxy)methyl)-3-((benzyloxy)methyl)-3-methyltetrahydrofuran-2,4-diyldibenzoate

To a solution of free alcohol (2.87 g, 5.85 mmol) in DCM/Cyclohexane(1:2) (60 mL, 0.1 M) at 0° C. was added benzyl2,2,2-trichloroacetimidate (3.26 mL, 17.55 mmol) and TfOH (51.6 μL,0.585 mmol) dropwise. After 16 hours at rt, the reaction was quenched byEt₃N (122 μL, 0.878 mmol) at 0° C., stirred for 15 minutes andconcentrated in vacuo. The product was filtered over celite with DCM,concentrated in vacuo and the crude product was purified by flashchromatography on silica gel (Hexanes/EtOAc, 80:20) to give a mixture ofproducts in a ˜5:1 ratio (2.53 g, 75% yield). R_(f)=0.4 (Hexanes/EtOAc,70:30); Formula: C₃₅H₃₂O₈; MW: 580.63 g/mol; IR (neat) v_(max) 3065,3033, 2941, 2882, 1720 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 8.10 (d, J=7.4Hz, 2H_(a)), 8.06 (d, J=7.5 Hz, 4H_(b)), 8.00 (d, J=7.4 Hz, 2H_(a)),7.96 (d, J=7.6 Hz, 2H_(a)), 7.92 (d, J=7.5 Hz, 2H_(b)), 7.59 (t, J=7.3Hz, 3H_(a)), 7.52 (t, J=7.5 Hz, 3H_(b)), 7.50-7.29 (m, 20H_(a,b)),7.26-7.20 (m, 2H_(a,b)), 6.66 (s, 1H_(b)), 6.62 (s, 1H_(a)), 5.79 (d,J=6.6 Hz, 1H_(b)), 4.74 (s, 2H_(a)), 4.68 (d, J=11.6 Hz, 1H_(a)), 4.64(s, 2H_(b)), 4.63-4.49 (m, 6H_(a,b)), 4.44 (dd, J=11.8, 5.1 Hz, 1H_(a)),4.29 (d, J=7.2 Hz, 1H_(a)), 3.84 (d, J=9.3 Hz, 1H_(b)), 3.66 (d, J=9.2Hz, 1H_(b)), 1.41 (s, 3H_(a)), 1.38 (s, 3H_(b)) ppm; ¹³C NMR (125 MHz,CDCl₃) δ 166.4, 166.3, 165.3, 137.3, 133.4, 133.2, 133.1, 129.72 (3C),129.68 (3C), 129.67 (3C), 128.6 (2C), 128.53 (2C), 128.52 (2C), 128.3(2C), 128.2, 127.7 (2C), 101.0, 84.6, 81.6, 73.9, 65.9, 65.3, 50.0, 17.3ppm; HRMS calcd for: C₃₅H₃₂NaO₈ [M+Na]⁺: 603.1989; found 603.1975 (−2.34ppm).

Example 2.28—Intermediate Compound

((2R,3S,4R,5R)-3-(benzoyloxy)-4-((benzyloxy)methyl)-5-(2,6-dichloro-9H-purin-9-yl)-4-methyltetrahydrofuran-2-yl)methylbenzoate (β-anomer)

To a mixture of anomeric benzoates (2.53 g, 4.36 mmol) and2,6-dichloropurine (907 mg, 4.79 mmol) in dry acetonitrile (22 mL, 0.2M) at −10° C. was added DBU (1.99 mL, 13.1 mmol). The mixture wasstirred and then TMSOTf (3.21 mL, 17.4 mmol) was added dropwise over 2minutes. After 5 hours at −10° C., the reaction was quenched by additionof a saturated solution of NaHCO₃ at −10° C., suspended in DCM andwashed with a saturated solution of NaHCO₃. The aqueous layer wasextracted with DCM (3×) and the combined organic fractions were washedwith an aqueous solution of citric acid (0.1 M), dried (MgSO₄), filteredand concentrated in vacuo. ¹H-NMR of the crude showed a ˜7:1 ratio ofβ:α anomers. Crude product was purified by flash chromatography onsilica gel (Hexanes/EtOAc, 70:30) to provide the pure β-anomer (1.08 g,40% yield). R_(f)=0.25 (Hexanes/EtOAc, 70:30); [α]²⁵ _(D) +16 (c 0.76,MeOH); Formula: C₃₃H₂₈Cl₂N₄O₆; MW: 647.51 g/mol; IR (neat) v_(max) 3124,3065, 2920, 1719, 1268 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 8.37 (s, 1H),8.04 (dd, J=8.4, 1.3 Hz, 2H), 7.91 (dd, J=8.3, 1.4 Hz, 2H), 7.65 (t,J=7.5 Hz, 1H), 7.60 (t, J=7.5 Hz, 1H), 7.52 (appt, J=7.8 Hz, 2H), 7.45(appt, J=7.8 Hz, 2H), 7.34 (d, J=4.4 Hz, 4H), 7.33-7.29 (m, 1H), 6.54(s, 1H), 4.79 (d, J=11.7 Hz, 1H), 4.71 (d, J=5.5 Hz, 2H), 4.70-4.67 (m,2H), 4.57 (d, J=11.7 Hz, 1H), 4.55-4.51 (m, 1H), 4.24 (d, J=6.8 Hz, 1H),1.03 (s, 3H) ppm; ¹³C NMR (125 MHz, CDCl₃) δ 166.2, 166.1, 153.2, 152.4,152.0, 144.0, 136.6, 133.6, 133.4, 130.9, 129.6 (3C), 129.4 (3C), 129.3,128.8 (2C), 128.7 (2C), 128.6 (2C), 128.2 (2C), 89.1, 82.9, 81.1, 73.6,66.3, 63.7, 49.4, 17.9 ppm; HRMS calcd for: C₃₃H₂₈Cl₂N₄NaO₆ [M+Na]⁺:669.1278; found 669.1272 (−0.98 ppm).

Example 2.29—Intermediate Compound

(2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-4-((benzyloxy)methyl)-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol

To a solution of protected nucleoside (371 mg, 0.573 mmol) in MeOH (12mL, 0.05 M) in a high-pressure flask at room temperature was bubbledNH_(3(g)) until saturation of the system, then the flask was rapidlyclosed with a high-pressure seal and the solution was stirred at 80° C.for 24 hours. NH_(3(g)) was bubbled for a second time until saturationof the system and after closing the flask with the high-pressure seal,the reaction was stirred for another 16 hours at 80° C. The mixture wasdiluted with MeOH (10 mL) and concentrated in vacuo. Crude product waspurified by flash chromatography on silica gel (DCM/MeOH, 90:10) to givethe product as a white solid (178 mg, 74% yield). R_(f)=0.4 (DCM/MeOH,90:10); [α]²⁵ _(D) −13 (c 1.13, MeOH); Formula: C₁₉H₂₂ClN₅O₄; MW: 419.87g/mol; IR (neat) v_(max) 3357, 2936, 2882, 2362, 1614 cm⁻¹; ¹H NMR (500MHz, CD₃OD) δ 8.45 (s, 1H), 7.41 (d, J=6.8 Hz, 2H), 7.36 (appt, J=7.4Hz, 2H), 7.30 (t, J=7.2 Hz, 1H), 6.27 (s, 1H), 4.76-4.66 (m, 2H), 4.39(d, J=8.3 Hz, 1H), 4.20 (ddd, J=8.3, 3.6, 2.5 Hz, 1H), 3.97 (dd, J=12.4,2.4 Hz, 1H), 3.91 (d, J=11.3 Hz, 1H), 3.86 (dd, J=12.4, 3.7 Hz, 1H),3.77 (d, J=11.3 Hz, 1H), 0.68 (s, 3H) ppm (Labile protons were notobserved due to exchange with deuterated solvent); ¹³C NMR (125 MHz,CD₃OD) δ 156.7, 153.9, 150.1, 140.5, 138.1, 128.0 (2C), 127.9 (2C),127.6, 117.6, 89.2, 83.6, 82.4, 73.4, 64.0, 60.8, 50.6, 16.5 ppm; HRMScalcd for: C₁₉H₂₃ClN₅O₄[M+H]⁺: 420.1433; found 420.1429 (−0.90 ppm).

Example 2.30—Intermediate Compound

9-((2R,3R,4S,5R)-3-((benzyloxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3-methyltetrahydrofuran-2-yl)-2-chloro-9H-purin-6-amine

To a solution of nucleoside (317 mg, 0.756 mmol) in DCM (7.6 mL, 0.1 M)and DMF (1 drop) at 0° C. was added 2,6-lutidine (438 μL, 3.78 mmol) andTBSOTf (0.515 mL, 2.27 mmol) dropwise. The reaction was stirred for 16hours at room temperature, then quenched by addition of a saturatedsolution of NaHCO₃ and concentrated in vacuo. The product was dilutedwith Et₂O, washed with brine and HCl [0.1 M], dried (MgSO₄), filteredand concentrated in vacuo. Crude product was purified by flashchromatography on silica gel (Hexanes/EtOAc, 60:40) to give the productas a white foam (321 mg, 65% yield). R_(f)=0.3 (Hexanes/EtOAc, 60:40);[α]²⁵ _(D) +7 (c 1.42, MeOH); Formula: C₃₁H₅₀ClN₅O₄Si₂; MW: 648.39g/mol; IR (neat) v_(max) 2925, 2855, 2324, 1612 cm⁻¹; ¹H NMR (500 MHz,CD₃OD) δ 8.48 (s, 1H), 7.36 (d, J=4.4 Hz, 4H), 7.34-7.28 (m, 1H), 6.30(s, 1H), 4.68 (s, 2H), 4.31 (d, J=8.1 Hz, 1H), 4.23 (dt, J=8.2, 3.0 Hz,1H), 4.04 (dd, J=11.7, 2.5 Hz, 1H), 4.01 (d, J=10.0 Hz, 1H), 3.95 (dd,J=11.7, 3.2 Hz, 1H), 3.78 (d, J=10.1 Hz, 1H), 0.98 (s, 9H), 0.95 (s,9H), 0.70 (s, 3H), 0.15 (s, 3H), 0.15 (s, 3H), 0.12 (s, 6H) ppm (Labileprotons were not observed due to exchange with deuterated solvent); ¹³CNMR (125 MHz, CD₃OD) δ 156.6, 154.1, 150.2, 140.0, 138.1, 128.1 (2C),127.62, 127.60 (2C), 117.4, 88.5, 83.5, 82.1, 73.4, 64.9, 62.3, 50.8,25.2 (3C), 25.1 (3C), 18.0, 17.8, 16.8, −6.59, −6.61, −6.7, −6.9 ppm;HRMS calcd for: C₃₁H₅₁ClN₅O₄Si₂ [M+H]⁺: 648.3163; found 648.3175 (1.98ppm).

Example 2.31—Intermediate Compound

((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-4-((benzyloxy)methyl)-3-((tert-butyldimethylsilyl)oxy)-4-methyltetrahydrofuran-2-yl)methanol

To a solution of nucleoside (358 mg, 0.552 mmol) in THF (6.1 mL, 0.09 M)at 0° C. was added TFA/H₂O (1:1, 3.08 mL, 0.18 M) and the mixture wasstirred for 4 hours at 0° C., then quenched slowly with a saturatedsolution of NaHCO₃. Aqueous phase was extracted with Et₂O (2×) and theorganic fractions were washed with brine, dried (MgSO₄), filtered andconcentrated in vacuo. Crude product was purified by flashchromatography on silica gel (DCM/MeOH, 95:5) to give the product as awhite solid (207 mg, 70% yield). R_(f)=0.5 (DCM/MeOH, 95:5); [α]²⁵ _(D)−14 (c 0.62, MeOH); Formula: C₂₅H₃₆ClN₅O₄Si; MW: 534.13 g/mol; IR (neat)v_(max) 2931, 2850, 2566, 2313, 1613 cm⁻¹; ¹H NMR (500 MHz, CD₃OD) δ8.49 (s, 1H), 7.42-7.33 (m, 4H), 7.33-7.28 (m, 1H), 6.31 (s, 1H),4.79-4.60 (m, 2H), 4.32 (d, J=8.0 Hz, 1H), 4.20 (dt, J=8.1, 3.0 Hz, 1H),4.01 (d, J=10.0 Hz, 1H), 3.97 (dd, J=12.4, 2.5 Hz, 1H), 3.84 (dd,J=12.4, 3.5 Hz, 1H), 3.77 (d, J=10.0 Hz, 1H), 0.94 (s, 9H), 0.69 (s,3H), 0.11 (s, 6H) ppm (Labile protons were not observed due to exchangewith deuterated solvent); ¹³C NMR (125 MHz, CD₃OD) δ 156.6, 154.0,150.2, 140.4, 138.2, 128.0 (2C), 127.7 (2C), 127.5, 117.4, 88.8, 83.6,82.2, 73.4, 65.0, 60.7, 50.7, 25.0 (3C), 17.8, 16.7, −6.7, −6.9 ppm;HRMS calcd for: C₂₅H₃₇ClN₅O₄Si [M+H]⁺: 534.2298; found 534.2290 (−1.40ppm).

Example 2.32—Intermediate Compound

((2R,3R,4S,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-4-((benzyloxy)methyl)-3-((tert-butyldimethylsilyl)oxy)-4-methyltetrahydrofuran-2-yl)methyldiethyl phosphate

To a solution of nucleoside (200 mg, 0.374 mmol) in THF (4 mL, 0.1 M)was added t-BuMgCl (0.562 mL, 0.562 mmol) dropwise at room temperatureand the mixture was stirred for 45 minutes. Diethylchlorophosphate (81.3μL, 0.562 mmol) was added slowly and the reaction was stirred for 16hours. The reaction was quenched by addition of MeOH and concentrated invacuo. Crude product was purified by reverse phase (C18) flashchromatography (H₂O→MeCN) to give the product as a white foam (216 mg,86% yield). R_(f)=0.5 (DCM/MeOH, 95:5); [α]²⁵ _(D) +1 (c 0.78, MeOH);Formula: C₂₉H₄₅ClN₅O₇PSi; MW: 670.22 g/mol; IR (neat) v_(max) 3172,2931, 2855, 2362, 1642, 1583 cm⁻¹; ¹H NMR (500 MHz, CD₃OD) δ 8.18 (s,1H), 7.45-7.36 (m, 4H), 7.36-7.30 (m, 1H), 6.28 (s, 1H), 4.71 (d, J=3.2Hz, 2H), 4.44-4.38 (m, 1H), 4.37 (s, 2H), 4.27 (dd, J=10.6, 5.7 Hz, 1H),4.17-4.05 (m, 4H), 4.03 (d, J=10.1 Hz, 1H), 3.76 (d, J=10.1 Hz, 1H),1.30 (qt, J=7.1, 1.1 Hz, 6H), 0.95 (s, 9H), 0.73 (s, 3H), 0.13 (s, 6H)ppm (Labile protons were not observed due to exchange with deuteratedsolvent); ¹³C NMR (125 MHz, CD₃OD) δ 156.7, 154.0, 150.1, 140.2, 137.9,128.2 (2C), 127.9 (2C), 127.7, 117.7, 89.3, 83.0, 81.6 (d, J=7.6 Hz),73.5, 67.0 (d, J=5.8 Hz), 65.1, 64.2 (t, J=5.4 Hz) (2C), 50.5, 25.0(3C), 17.7, 16.6, 15.0 (t, J=5.8 Hz) (2C), −6.7, −6.9 ppm; HRMS calcdfor: C₂₉H₄₅ClN₅NaO₇PSi [M+Na]⁺: 692.2407; found 692.2419 (1.78 ppm).

Example 2.33—Cardioprotective Compound-LCB2165

((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-4-((benzyloxy)methyl)-3-hydroxy-4-methyltetrahydrofuran-2-yl)methyldiethyl phosphate (LCB2165)

To a solution of TBS protected nucleoside (29.3 mg, 0.0437 mmol) in THF(0.5 mL, 0.1 M) was slowly added a mixture of TFA/H₂O (8:2) (0.5 mL, 0.1M) at room temperature and open atmosphere. After 4 hours, MeOH wasadded and the reaction was concentrated in vacuo, then co-evaporatedwith MeOH (3×). Crude product was purified by reverse phase (C18) flashchromatography (H₂O/MeCN, 50:50) to provide the final product as a whitesolid (10.9 mg, 45% yield). R_(f)=0.4 (DCM/MeOH, 90:10); [α]²⁵ _(D) +8(c 1.02, MeOH); Formula: C₂₃H₃₁ClN₅O₇P; MW: 555.95 g/mol; IR (neat)v_(max) 3370, 2979, 2909, 2383, 1613 cm⁻¹; ¹H NMR (500 MHz, CD₃OD) δ8.17 (s, 1H), 7.43 (d, J=6.8 Hz, 2H), 7.39 (appt, J=7.2 Hz, 2H),7.36-7.31 (m, 1H), 6.25 (s, 1H), 4.77-4.64 (m, 2H), 4.50-4.40 (m, 2H),4.38-4.32 (m, 1H), 4.25 (ddd, J=11.1, 5.7, 2.5 Hz, 1H), 4.17-4.02 (m,4H), 3.93 (d, J=11.2 Hz, 1H), 3.77 (d, J=11.2 Hz, 1H), 1.28 (dtd,J=10.7, 7.1, 1.0 Hz, 6H), 0.74 (s, 3H) ppm (Labile protons were notobserved due to exchange with deuterated solvent); ¹³C NMR (125 MHz,CD₃OD) δ 156.8, 153.9, 149.9, 140.5, 137.9, 128.2 (2C), 128.1 (2C),127.8, 117.9, 89.6, 83.0, 81.6 (d, J=7.7 Hz), 73.5, 67.1 (d, J=5.8 Hz),64.2 (t, J=5.7 Hz) (2C), 64.0, 50.4, 16.4, 15.0 (t, J=5.8 Hz) (2C) ppm;HRMS calcd for: C₂₃H₃₂ClN₅O₇P [M+H]⁺: 556.1722; found 556.1748 (4.61ppm).

Example 2.34—Intermediate Compound

(4R,5R)-3-bromo-4-hydroxy-5-(((4-methoxybenzyl)oxy)methyl)-3-methyldihydrofuran-2(3H)-one

To a solution of lactones (2.01 g, 8.92 mmol) in DCM/Cyclohexane (1:2,90 mL, 0.1 M) with DMF (˜3 mL) at 0° C. was added4-methoxybenzyl-2,2,2-trichloroacetimidate (2.04 mL, 9.81 mmol) and TfOH(78.7 μL, 0.892 mmol) dropwise. The mixture was warmed to roomtemperature, stirred for 16 hours and quenched with Et₃N (187 μL, 1.34mmol) at 0° C. After 10 minutes, the reaction was concentrated in vacuoand the crude product was purified by flash chromatography on silica gel(Hexanes/Et₂O, 30:70) to give the products (2.60 g, 84% yield) as a paleyellow solid in a ˜16:1 ratio. R_(f)=0.25 (Hexanes/Et₂O, 30:70);Formula: C₁₄H₁₇BrO₅; MW: 345.19 g/mol; IR (neat) v_(max) 3435, 2931,2866, 1782, 1513 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 7.26 (d, J=8.6 Hz,4H_(a,b)), 6.90 (d, J=8.6 Hz, 4H_(a,b)), 4.71 (d, J=6.0 Hz, 2H_(b)),4.53 (d, J=2.0 Hz, 2H_(a)), 4.35 (dt, J=5.9, 4.9 Hz, 1H_(b)), 4.25 (ddd,J=8.3, 4.0, 2.8 Hz, 1H_(a)), 3.85 (dd, J=11.5, 2.8 Hz, 2H_(a)), 3.82 (s,6H_(a,b)), 3.80-3.77 (m, 3H_(b)), 3.74 (dd, J=11.5, 4.0 Hz, 1H_(a)),1.95 (s, 3H_(a)), 1.87 (s, 3H_(b)) ppm (Labile protons were not observeddue to exchange with deuterated solvent); ¹³C NMR (125 MHz, CDCl₃) δ171.5, 159.5, 129.5 (2C), 129.3, 113.9 (2C), 81.5, 74.3, 73.4, 66.1,61.7, 55.3, 24.0 ppm; HRMS calcd for: C₁₄H₁₇BrNaO₅ [M+Na]⁺: 367.0152;found 367.0151 (−0.21 ppm).

Example 2.35—Intermediate Compound

(4R,5R)-3-bromo-4-((dimethyl(vinyl)silyl)oxy)-5-(((4-methoxybenzyl)oxy)methyl)-3-methyldihydrofuran-2(3H)-one

To a solution of lactones (2.60 g, 7.53 mmol) in DCM (75 mL, 0.1 M) at0° C. was added pyridine (1.83 mL, 22.6 mmol) andchloro(dimethyl)vinylsilane (1.13 mL, 8.29 mmol). The reaction wasstirred at room temperature for 16 hours, then concentrated in vacuo.Precipitate was removed by filtration over celite with Et₂O and crudeproduct was purified by flash chromatography on silica gel(Hexanes/Et₂O, 60:40) to give the two products (2.63 g, 81% yield) as acolorless oil in a ˜3:1 ratio. R_(f)=0.5 (Hexanes/Et₂O, 50:50); Formula:C₁₈H₂₅BrO₅Si; MW: 429.38 g/mol; IR (neat) v_(max) 2947, 2866, 1793, 1513cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 7.26 (d, J=8.6 Hz, 4H_(a,b)), 6.91 (d,J=8.7 Hz, 4H_(a,b)), 6.17-6.05 (m, 4H_(a,b)), 5.85 (dd, J=17.4, 6.6 Hz,1H_(a)), 5.82 (dd, J=18.9, 5.1 Hz, 1H_(b)), 4.73 (d, J=5.8 Hz, 1H_(b)),4.54 (s, 2H_(b)), 4.51 (d, J=1.5 Hz, 2H_(a)), 4.33-4.24 (m, 2H_(a,b)),3.94 (d, J=8.1 Hz, 1H_(a)), 3.83 (s, 6H_(a,b)), 3.81 (d, J=2.0 Hz,1H_(a)), 3.78 (dd, J=11.2, 4.0 Hz, 1H_(b)), 3.68 (dd, J=11.2, 4.9 Hz,1H_(b)), 3.63 (dd, J=11.8, 3.2 Hz, 1H_(a)), 1.87 (s, 3H_(a)), 1.82 (s,3H_(b)), 0.28 (s, 3H_(b)), 0.27 (s, 6H_(a,b)), 0.26 (s, 3H_(a)) ppm; ¹³CNMR (125 MHz, CDCl₃) δ 173.2, 172.1, 159.4, 136.2, 136.1, 134.8, 134.7,129.52 (2C), 129.46, 113.9 (2C), 83.1, 81.3, 78.2, 74.1, 73.3, 73.2,67.2, 65.2, 59.9, 56.8, 55.3, 24.5, 22.1, −1.4, −1.58, −1.62, −1.7 ppm;HRMS calcd for: C₁₈H₂₅BrNaO₅Si [M+Na]⁺: 451.0547; found 451.0545 (−0.51ppm).

Example 2.36—Intermediate Compound

(3R,4S,5R)-4-hydroxy-5-(((4-methoxybenzyl)oxy)methyl)-3-methyl-3-vinyldihydroofuran-2(3H)-one

To a solution of lactones (1.15 g, 2.68 mmol) in toluene (27 mL, 0.1 M)at 0° C. with open atmosphere was added triethylborane (2.68 mL, 2.68mmol) over 5 hours with air bubbling into the solution. The reaction wastransferred into a plastic flask, then 3HF.NEt₃ (873 μL, 5.36 mmol) andTHF (27 mL, 0.1 M) were added at 0° C. The mixture was stirred for 16hours at room temperature and quenched slowly by cannulation into asaturated solution of NaHCO₃ at 0° C. Aqueous phase was extracted withEt₂O (2×) and organic fractions were dried (MgSO₄), filtered andconcentrated in vacuo. Crude product was purified by flashchromatography on silica gel (Hexanes/Et₂O, 40:60) to give the product(475 mg, 61% yield) as a colorless oil. R_(f)=0.3 (Hexanes/Et₂O, 20:80);[α]²⁵ _(D) +57 (c 0.57, MeOH); Formula: C₁₆H₂₀O₅; MW: 292.33 g/mol; IR(neat) v_(max) 3435, 2935, 2866, 1771, 1513 cm⁻¹; ¹H NMR (500 MHz,CDCl₃) δ 7.27 (d, J=8.6 Hz, 2H), 6.91 (d, J=8.6 Hz, 2H), 5.93 (dd,J=17.6, 10.7 Hz, 1H), 5.39 (d, J=10.7 Hz, 1H), 5.27 (d, J=17.6 Hz, 1H),4.54 (d, J=1.4 Hz, 2H), 4.21 (dt, J=8.8, 4.4 Hz, 1H), 4.10 (d, J=8.4 Hz,1H), 3.83 (s, 3H), 3.76 (dd, J=4.5, 2.3 Hz, 2H), 1.41 (s, 3H) ppm(Labile protons were not observed due to exchange with deuteratedsolvent); ¹³C NMR (125 MHz, CDCl₃) δ 176.2, 159.5, 132.9, 129.5 (2C),129.4, 118.4, 113.9 (2C), 79.8, 77.1, 73.5, 68.2, 55.3, 51.1, 20.5 ppm;HRMS calcd for: C₁₆H₂₀NaO₅ [M+Na]⁺: 315.1203; found 315.1199 (−1.15ppm).

Example 2.37—Intermediate Compound

(3R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((4-methoxybenzyl)oxy)methyl)-3-methyl-3-vinyldihydrofuran-2(3H)-one

To a solution of lactone (1.71 g, 5.84 mmol) in DCM (60 mL, 0.1 M) at 0°C. was added 2,6-lutidine (1.69 mL, 14.6 mmol) and TBSOTf (2.65 mL, 11.7mmol) dropwise. The reaction was stirred at room temperature for 16hours, then quenched by slowly adding a saturated solution of NaHCO₃ andconcentrated in vacuo. The product was diluted with Et₂O, washed withbrine and HCl [0.1 M], dried (MgSO₄), filtered and concentrated invacuo. Crude product was purified by flash chromatography on silica gel(Hexanes/Et₂O, 60:40) to give the product (1.87 g, 79% yield) as ayellow oil. R_(f)=0.3 (Hexanes/Et₂O, 60:40); [α]²⁵ _(D) +56 (c 1.11,MeOH); Formula: C₂₂H₃₄O₅Si; MW: 406.59 g/mol; IR (neat) v_(max) 2958,2931, 2855, 1787, 1513 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 7.27 (d, J=9.7Hz, 2H), 6.90 (d, J=8.6 Hz, 2H), 5.96 (dd, J=17.6, 10.7 Hz, 1H), 5.28(d, J=10.7 Hz, 1H), 5.22 (d, J=17.7 Hz, 1H), 4.52 (s, 2H), 4.18 (d,J=8.4 Hz, 1H), 4.14 (ddd, J=8.4, 3.8, 1.9 Hz, 1H), 3.83 (s, 3H), 3.79(dd, J=11.6, 1.9 Hz, 1H), 3.59 (dd, J=11.6, 3.8 Hz, 1H), 1.36 (s, 3H),0.90 (s, 9H), 0.11 (s, 3H), 0.05 (s, 3H) ppm; ¹³C NMR (125 MHz, CDCl₃) δ176.3, 159.3, 133.8, 129.6, 129.5 (2C), 116.9, 113.8 (2C), 81.1, 76.0,73.2, 66.6, 55.3, 51.4, 25.6 (3C), 21.0, 17.9, −4.3, −4.8 ppm; HRMScalcd for: C₂₂H₃₄NaO₅Si [M+Na]⁺: 429.2068; found 429.2071 (0.84 ppm).

Example 2.38—Intermediate Compound

(3S,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((4-methoxybenzyl)oxy)methyl)-3-methyl-2-oxotetrahydrofuran-3-carbaldehyde

To a solution of lactone (1.78 g, 4.38 mmol) in DCM (88 mL, 0.05 M) wasadded a pinch of Sudan Red 7B and the reaction (red/purple solution) wascooled to −78° C. Ozone was bubbled into the solution for 30 minutes(solution turned orange), then Et₃N (1.22 mL, 8.76 mmol) was added, themixture was warmed to room temperature, stirred for 30 minutes andconcentrated in vacuo. The product was filtered over silica with Et₂O togive the desired aldehyde (1.77 g, quantitative yield) as an orange oilthat was used for the next reaction without further purification.R_(f)=0.55 (Hexanes/Et₂O, 20:80); [α]²⁵ _(D) +41 (c 1.16, MeOH);Formula: C₂₁H₃₂O₆Si; MW: 408.57 g/mol; IR (neat) v_(max) 2958, 2931,2855, 1787, 1723, 1508 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 9.58 (s, 1H),7.25 (d, J=8.7 Hz, 2H), 6.90 (d, J=8.7 Hz, 2H), 4.54 (d, J=11.6 Hz, 1H),4.50 (d, J=11.6 Hz, 1H), 4.48-4.42 (m, 2H), 3.83 (s, 3H), 3.78 (dd,J=11.5, 2.2 Hz, 1H), 3.62 (dd, J=11.5, 2.8 Hz, 1H), 1.51 (s, 3H), 0.86(s, 9H), 0.10 (s, 3H), 0.03 (s, 3H) ppm; ¹³C NMR (125 MHz, CDCl₃) δ195.8, 172.7, 159.5, 129.6 (2C), 129.3, 113.9 (2C), 82.8, 77.6, 73.4,66.3, 60.1, 55.3, 25.5 (3C), 17.8, 15.3, −4.6, −4.9 ppm; HRMS calcd for:C₂₁H₃₂NaO₆Si [M+Na]⁺: 431.1860; found 431.1860 (−0.074 ppm).

Example 2.39—Intermediate Compound

(3R,4S,5R)-4((tert-butyldimethylsilyl)oxy)-3-(hydroxymethyl)-5-(((4-methoxybenzyl)oxy)methyl)-3-methyldihydrofuran-2(3H)-one

To a solution of aldehyde (1.77 g, 4.33 mmol) in THF (44 mL, 0.1 M) at−40° C. was added LiAlH(Ot-Bu)₃ (6.50 mL, 6.50 mmol) dropwise and thereaction was stirred for 2 hours at −40° C. Sodium sulfate decahydrate(2.79 g, 8.66 mmol) was added and the mixture was warmed to roomtemperature, stirred for 45 minutes and concentrated in vacuo. Theproduct was filtered over silica with Et₂O and was obtained as an orangeoil that was used for the next reaction without further purification(1.25 g, 70% yield). R_(f)=0.45 (Hexanes/Et₂O, 20:80); [α]²⁵ _(D) +38 (c2.05, MeOH); Formula: C₂₁H₃₄O₆Si; MW: 410.58 g/mol; IR (neat) v_(max)3494, 2929, 2857, 1773, 1513, 1249 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 7.25(d, J=8.4 Hz, 2H), 6.90 (d, J=8.4 Hz, 2H), 4.55 (d, J=11.6 Hz, 1H), 4.49(d, J=11.7 Hz, 1H), 4.38 (d, J=5.9 Hz, 1H), 4.35-4.30 (m, 1H), 3.83 (s,3H), 3.81 (d, J=4.6 Hz, 1H), 3.79-3.71 (m, 2H), 3.61 (dd, J=11.3, 3.0Hz, 1H), 1.19 (s, 3H), 0.92 (s, 9H), 0.14 (s, 3H), 0.07 (s, 3H) ppm(Labile protons were not observed due to exchange with deuteratedsolvent); ¹³C NMR (125 MHz, CDCl₃) δ 178.7, 159.4, 129.52 (2C), 129.47,113.9 (2C), 83.7, 76.9, 73.3, 67.4, 66.4, 55.3, 49.8, 25.6 (3C), 18.8,17.8, −4.5, −4.8 ppm; HRMS calcd for: C₂₁H₃₄NaO₆Si [M+Na]⁺: 433.2017;found 433.2017 (0.14 ppm).

Example 2.40—Intermediate Compound

((3R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((4-methoxybenzyl)oxy)methyl)-3-methyl-2-oxotetrahydrofuran-3-yl)methylbenzoate

To a solution of alcohol (1.21 g, 2.95 mmol) in DCM (30 mL, 0.1 M) wasadded DMAP (36 mg, 0.295 mmol) and pyridine (1.19 mL, 14.7 mmol) at 25°C. The mixture was cooled to 0° C. and benzoyl chloride (0.856 mL, 7.37mmol) was added. The reaction was warmed to room temperature and stirredfor 24 hours, then ethylenediamine (0.395 mL, 5.89 mmol) was addedslowly at 0° C. and the resultant mixture was stirred for 45 minutes at0° C. After concentration in vacuo, the mixture was filtered over celitewith Et₂O to remove the precipitate. The resultant oil was filtered oversilica with Et₂O and the desired product (1.59 g, quantitative yield)was obtained as a yellow oil that was used for the next reaction withoutfurther purification. R_(f)15=0.5 (Hexanes/Et₂O, 30:70); [α]²⁵ _(D) +40(c 1.20, MeOH); Formula: C₂₈H₃₈O₇Si; MW: 514.69 g/mol; IR (neat) v_(max)2953, 2931, 2857, 1782, 1725, 1268 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 8.00(d, J=7.9 Hz, 2H), 7.58 (t, J=7.5 Hz, 1H), 7.45 (appt, J=7.6 Hz, 2H),7.27 (d, J=8.3 Hz, 2H), 6.90 (d, J=8.3 Hz, 2H), 4.70 (d, J=10.8 Hz, 1H),4.54 (d, J=6.6 Hz, 2H), 4.41-4.33 (m, 2H), 4.18 (d, J=10.8 Hz, 1H), 3.83(s, 3H), 3.79 (d, J=11.7 Hz, 1H), 3.60 (dd, J=11.7, 3.0 Hz, 1H), 1.35(s, 3H), 0.84 (s, 9H), 0.11 (s, 3H), 0.02 (s, 3H) ppm; ¹³C NMR (125 MHz,CDCl₃) δ 177.0, 165.5, 159.4, 133.1, 129.7, 129.54 (3C), 129.50 (2C),128.5 (2C), 113.9 (2C), 82.8, 74.8, 73.3, 66.9, 65.4, 55.3, 47.4, 25.6(3C), 19.1, 17.8, −4.2, −4.9 ppm; HRMS calcd for: C₂₈H₃₉O₇Si [M+H]⁺:515.2460; found 515.2460 (0.0039 ppm).

Example 2.41—Intermediate Compound

((3R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-2-hydroxy-5-(((4-methoxybenzyl)oxy)methyl)-3-methyltetrahydrofuran-3-yl)methylbenzoate

To a solution of lactone (1.59 g, 3.09 mmol) in THF (31 mL, 0.1 M) at 0°C. was added LiAlH(Ot-Bu)₃ (6.18 mL, 6.18 mmol) dropwise and thereaction was stirred for 24 hours at room temperature. Sodium sulfatedecahydrate (1.99 g, 6.18 mmol) was added and the mixture was stirredfor 45 minutes, then concentrated in vacuo. The product was filteredover silica with Et₂O and the desired products (1.26 g, 79% yield) wereobtained in a ˜3:1 ratio of anomers as a yellow oil that was used forthe next reaction without further purification. R_(f)=0.45(Hexanes/Et₂O, 30:70); Formula: C₂₈H₄₀O₇Si; MW: 516.71 g/mol; IR (neat)v_(max) 3435, 2952, 2925, 2855, 1717, 1513, 1272 cm⁻¹; ¹H NMR (500 MHz,CDCl₃) δ 8.06 (dd, J=8.3, 1.4 Hz, 4H_(a,b)), 7.57 (t, J=7.4 Hz,2H_(a,b)), 7.46 (appq, J=8.1 Hz, 4H_(a,b)), 7.29 (d, J=8.6 Hz,4H_(a,b)), 6.92 (d, J=8.7 Hz, 4H_(a,b)), 5.26 (s, 1H_(a)), 5.11 (s,1H_(b)), 4.65 (d, J=11.5 Hz, 1H_(a)), 4.56-4.48 (m, 3H_(a,b)), 4.40 (d,J=11.5 Hz, 2H_(a,b)), 4.35 (d, J=11.5 Hz, 2H_(a,b)), 4.30 (d, J=6.3 Hz,1H_(a)), 4.06 (dt, J=6.3, 2.5 Hz, 2H_(a,b)), 3.99 (d, J=2.6 Hz, 1H_(b)),3.83 (s, 6H_(a,b)), 3.65 (dd, J=10.3, 2.5 Hz, 1H_(a)), 3.62-3.56 (m,2H_(b)), 3.54 (dd, J=10.3, 2.6 Hz, 1H_(a)), 1.23 (s, 3H_(a)), 1.16 (s,3H_(b)), 0.89 (s, 18H_(a,b)), 0.09 (s, 3H_(a)), 0.06 (s, 3H_(b)), 0.05(s, 3H_(b)), −0.04 (s, 3H_(a)) ppm (Labile protons were not observed dueto exchange with deuterated solvent); ¹³C NMR (125 MHz, CDCl₃) δ 166.54,166.48, 159.6, 159.3, 133.0, 132.9, 130.2, 129.7 (2C), 129.6 (3C),129.5, 129.4 (3C), 129.1 (2C), 128.41 (2C), 128.38 (2C), 114.0 (2C),113.8 (2C), 104.0, 101.5, 86.2, 83.7, 79.0, 77.4, 73.3, 73.0, 69.8,68.6, 66.7, 65.3, 55.3 (2C_(a,b)), 50.9, 49.8, 25.7 (6C_(a,b)), 19.9,17.9, 17.8, 16.3, −4.4, −4.57, −4.61, −5.1 ppm; HRMS calcd for:C₂₈H₄₀NaO₇Si [M+Na]⁺: 539.2436; found 539.2437 (0.22 ppm).

Example 2.42—Intermediate Compound

((3R,4S,5R)-2-acetoxy-4-((tert-butyldimethylsilyl)oxy)-5-(((4-methoxybenzyl)oxy)methyl)-3-methyltetrahydrofuran-3-yl)methylbenzoate

To a solution of lactols (97.8 mg, 0.189 mmol) in pyridine (2 mL, 0.1 M)at 0° C. was added acetic anhydride (71.6 μL, 0.757 mmol). The reactionwas warmed to room temperature, stirred for 24 hours and concentrated invacuo. The product was diluted with EtOAc, washed with brine, dried(MgSO₄), filtered and concentrated in vacuo. Crude product was purifiedby flash chromatography on silica gel (Hexanes/Et₂O, 65:35) to give ayellow oil as a mixture of products (76.7 mg, 73% yield) in a ˜9:1 ratioof anomers. R_(f)=0.35 (Hexanes/Et₂O, 60:40); Formula: C₃₀H₄₂O₈Si; MW:558.74 g/mol; IR (neat) v_(max) 2952, 2931, 2850, 1744, 1723, 1513, 1272cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 8.07 (dd, J=8.3, 1.4 Hz, 2H_(a)),8.04-7.97 (m, 2H_(b)), 7.58 (t, J=7.4 Hz, 2H_(a,b)), 7.46 (appt, J=7.8Hz, 4H_(a,b)), 7.28 (d, J=8.5 Hz, 4H_(a,b)), 6.90 (d, J=8.7 Hz,4H_(a,b)), 6.32 (s, 1H_(a)), 6.14 (s, 1H_(b)), 4.71 (d, J=10.8 Hz,1H_(b)), 4.53 (s, 4H_(a,b)), 4.47 (d, J=11.5 Hz, 1H_(a)), 4.39 (d,J=11.5 Hz, 1H_(a)), 4.26 (d, J=7.3 Hz, 1H_(a)), 4.18 (d, J=10.8 Hz,1H_(b)), 4.09 (ddd, J=7.6, 5.0, 2.9 Hz, 2H_(a,b)), 3.98 (d, J=3.7 Hz,1H_(b)), 3.83 (s, 6H_(a,b)), 3.67 (dd, J=10.9, 2.9 Hz, 1H_(a)), 3.63(dd, J=4.6, 1.4 Hz, 1H_(b)), 3.60 (dd, J=11.5, 3.1 Hz, 1H_(b)), 3.54(dd, J=10.9, 5.0 Hz, 1H_(a)), 2.04 (s, 3H_(b)), 1.99 (s, 3H_(a)), 1.25(s, 3H_(b)), 1.20 (s, 3H_(a)), 0.90 (s, 9H_(a)), 0.84 (s, 9H_(b)), 0.13(s, 3H_(a)), 0.05 (s, 3H_(a)), 0.02 (s, 3H_(b)), 0.01 (s, 3H_(b)) ppm;¹³C NMR (125 MHz, CDCl₃) δ 170.1, 166.4, 159.2, 133.0, 129.6 (3C),129.5, 129.2 (2C), 128.4 (2C), 113.7 (2C), 100.0, 84.0, 77.5, 72.8,69.7, 66.0, 55.3, 49.5, 25.7 (3C), 21.2, 17.9, 16.1, −4.3, −4.6 ppm;HRMS calcd for: C₃₀H₄₂NaO₈Si [M+Na]⁺: 581.2541; found 581.2542 (0.082ppm).

Example 2.43—Intermediate Compound

((2R,3S,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-2-(2,6-dichloro-9H-purin-9-yl)-5-(((4-methoxybenzyl)oxy)methyl)-3-methyltetrahydrofuran-3-yl)methylbenzoate

To a mixture of anomeric acetates (1.95 g, 3.50 mmol) and2,6-dichloropurine (727 mg, 3.85 mmol) in dry acetonitrile (35 mL, 0.1M) at −10° C. was added DBU (915 μL, 6.12 mmol). The mixture was stirredand then TMSOTf (1.27 mL, 6.99 mmol) was added dropwise over 2 minutes.After 4 hours at −10° C., the reaction was warmed to room temperature,stirred for 2 hours and quenched by addition of a saturated solution ofNaHCO₃ at 0° C. The aqueous layer was extracted with DCM (3×) and thecombined organic fractions were dried (MgSO₄), filtered and concentratedin vacuo. ¹H-NMR of the crude showed a ˜18:1 ratio of β:α anomers. Crudeproduct was purified by flash chromatography on silica gel(Hexanes/Et₂O, 60:40) to provide the pure β-anomer (1.32 g, 55% yield)as a white foam. R_(f)=0.4 (Hexanes/Et₂O, 50:50); [α]²⁵ _(D) −14 (c0.54, MeOH); Formula: C₃₃H₄₀Cl₂N₄O₆Si; MW: 687.69 g/mol; IR (neat)v_(max) 3113, 2958, 2925, 2855, 1723, 1589, 1556, 1358, 1250 cm⁻¹; ¹HNMR (500 MHz, CDCl₃) δ 9.09 (s, 1H), 8.27 (dd, J=8.3, 1.4 Hz, 2H), 7.63(t, J=7.4 Hz, 1H), 7.55 (appt, J=7.5 Hz, 2H), 7.33 (d, J=8.7 Hz, 2H),6.95 (d, J=8.6 Hz, 2H), 6.63 (s, 1H), 4.64 (d, J=11.6 Hz, 1H), 4.63-4.57(m, 2H), 4.55 (d, J=8.6 Hz, 1H), 4.49 (d, J=11.7 Hz, 1H), 4.16 (dt,J=8.6, 2.0 Hz, 1H), 3.94 (dd, J=11.3, 2.0 Hz, 1H), 3.85 (s, 3H), 3.69(dd, J=11.3, 2.0 Hz, 1H), 0.92 (s, 9H), 0.86 (s, 3H), 0.09 (s, 3H), 0.05(s, 3H) ppm; ¹³C NMR (125 MHz, CDCl₃) δ 166.6, 159.7, 152.9, 152.7,151.7, 145.2, 133.3, 130.8, 130.0 (2C), 129.9 (2C), 129.6, 128.8, 128.7(2C), 114.1 (2C), 88.2, 83.1, 74.6, 73.5, 66.6, 66.1, 55.3, 50.1, 25.7(3C), 17.9, 17.3, −4.3, −4.7 ppm; HRMS calcd for: C₃₃H₄₀Cl₂N₄NaO₆Si[M+Na]⁺: 709.1986; found 709.1980 (−0.96 ppm).

Example 2.44—Intermediate Compound

((2R,3R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-2-(2,6-dichloro-9H-purin-9-yl)-5-(hydroxymethyl)-3-methyltetrahydrofuran-3-yl)methylbenzoate

To a solution of protected nucleoside (493 mg, 717 μmol) in DCM (15 mL,0.05 M) at room temperature with open atmosphere was added DDQ (488 mg,2.15 mmol) and the mixture was stirred for 16 hours. A few drops ofwater were added and the reaction was stirred for another 7 hours. Themixture was slowly poured into a saturated solution of NaHCO₃. Theaqueous layer was extracted with DCM (3×) and the combined organicfractions were washed with brine, dried (MgSO₄), filtered andconcentrated in vacuo. Crude product was purified by flashchromatography on silica gel (Hexanes/Et₂O, 40:60) to give the product(367 mg, 90% yield) as a white solid. R_(f)=0.2 (Hexanes/Et₂O, 50:50);[α]²⁵ _(D) −12 (c 1.67, MeOH); Formula: C₂₅H₃₂Cl₂N₄O₅Si; MW: 567.54g/mol; IR (neat) v_(max) 3317, 3102, 3065, 2925, 2855, 1723, 1594, 1551,1363 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 9.27 (s, 1H), 8.25 (dd, J=8.4, 1.4Hz, 2H), 7.64 (t, J=7.4 Hz, 1H), 7.55 (appt, J=7.6 Hz, 2H), 6.62 (s,1H), 4.66 (d, J=8.6 Hz, 1H), 4.63 (d, J=11.7 Hz, 1H), 4.52 (d, J=11.6Hz, 1H), 4.31 (ddd, J=12.2, 4.2, 2.0 Hz, 1H), 4.16 (dt, J=8.6, 2.0 Hz,1H), 4.06 (t, J=4.0 Hz, 1H), 3.97 (ddd, J=12.3, 3.9, 1.9 Hz, 1H), 0.96(s, 9H), 0.92 (s, 3H), 0.22 (s, 3H), 0.17 (s, 3H) ppm; ¹³C NMR (125 MHz,CDCl₃) δ 166.5, 153.1, 152.5, 151.6, 145.5, 133.4, 130.4, 129.8 (2C),129.5, 128.7 (2C), 88.8, 84.2, 74.2, 66.4, 59.4, 50.2, 25.7 (3C), 17.9,17.5, −4.2, −4.6 ppm; HRMS calcd for: C₂₅H₃₂Cl₂N₄NaO₅Si [M+Na]⁺:589.1411; found 589.1398 (−2.32 ppm).

Example 2.45—Intermediate Compound

((2R,3S,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-2-(2,6-dichloro-9H-purin-9-yl)-5-(((diethoxyphosphoryl)oxy)methyl)-3-methyltetrahydrofuran-3-yl)methylbenzoate

To a solution of nucleoside (349 mg, 615 μmol) in THF (6.15 mL, 0.1 M)was added t-BuMgCl (1.35 mL, 1.08 mmol) dropwise at room temperature andthe mixture was stirred for 45 minutes. Diethylchlorophosphate (156 μL,1.08 mmol) was added slowly and the reaction was stirred for 4 hours.The reaction was quenched by addition of MeOH and concentrated in vacuo.Crude product was purified by flash chromatography on silica gel(Hexanes/EtOAc, 50:50) to give the product (389 mg, 90% yield) as ayellow oil. R_(f)=0.35 (Hexanes/EtOAc, 40:60); [α]²⁵ _(D) +10 (c 1.36,MeOH); Formula: C₂₉H₄₁Cl₂N₄O₈PSi; MW: 703.63 g/mol; IR (neat) v_(max)2953, 2931, 2850, 1723, 1589, 1551, 1358, 1267 cm⁻¹; ¹H NMR (500 MHz,CDCl₃) δ 8.62 (s, 1H), 8.11 (dd, J=8.3, 1.4 Hz, 2H), 7.62 (t, J=7.4 Hz,1H), 7.51 (appt, J=7.8 Hz, 2H), 6.62 (s, 1H), 4.65 (d, J=11.7 Hz, 1H),4.52 (ddd, J=11.8, 4.7, 2.2 Hz, 1H), 4.49-4.43 (m, 2H), 4.32 (ddd,J=11.8, 4.5, 2.8 Hz, 1H), 4.26-4.17 (m, 5H), 1.43-1.35 (m, 6H), 0.95 (s,9H), 0.94 (s, 3H), 0.20 (s, 3H), 0.17 (s, 3H) ppm; ¹³C NMR (125 MHz,CDCl₃) δ 166.3, 153.1, 152.6, 151.9, 144.4, 133.4, 130.8, 129.6 (2C),129.4, 128.7 (2C), 88.5, 82.5 (d, J=8.6 Hz), 75.5, 66.3, 64.5 (d, J=4.8Hz), 64.4 (d, J=5.8 Hz), 64.3 (d, J=5.7 Hz), 49.8, 25.7 (3C), 17.9,17.4, 16.3 (d, J=4.9 Hz), 16.2 (d, J=5.1 Hz), −4.3, −4.5 ppm; HRMS calcdfor: C₂₉H₄₁Cl₂N₄NaO₈PSi [M+Na]⁺: 725.1701; found 725.1688 (−1.73 ppm).

Example 2.46—Intermediate Compound

((2R,3R,4S,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-3-((tert-butyldimethylsilyl)oxy)-4-(hydroxymethyl)-4-methyltetrahydrofuran-2-yl)methyldiethyl phosphate

To a solution of nucleoside (596 mg, 847 μmol) in MeOH (21 mL, 0.04 M)in a high-pressure flask at room temperature was bubbled NH_(3(g)) untilsaturation of the system, then the flask was rapidly closed with ahigh-pressure seal and the solution was stirred at 80° C. for 24 hours.The mixture was diluted with MeOH (20 mL), cooled to room temperatureand concentrated in vacuo. Crude product was purified by flashchromatography on silica gel (DCM/MeOH, 95:5) to give the product (251mg, 51% yield) as a white solid. R_(f)=0.15 (DCM/MeOH, 95:5); [α]²⁵ _(D)+2 (c 1.06, MeOH); Formula: C₂₂H₃₉ClN₅O₇PSi; MW: 580.09 g/mol; IR (neat)v_(max) 3296, 2952, 2928, 2855, 1613 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ8.11 (s, 1H), 6.30 (s, 1H), 5.77 (s, 2H), 4.44 (ddd, J=11.5, 5.7, 2.3Hz, 1H), 4.40 (d, J=7.3 Hz, 1H), 4.31 (dt, J=11.2, 5.1 Hz, 1H),4.24-4.12 (m, 5H), 3.94 (dd, J=11.8, 5.6 Hz, 1H), 3.85 (dd, J=11.8, 7.6Hz, 1H), 2.67 (dd, J=7.5, 5.6 Hz, 1H), 1.37 (tdd, J=7.1, 3.2, 1.0 Hz,6H), 0.96 (s, 9H), 0.76 (s, 3H), 0.20 (s, 3H), 0.17 (s, 3H) ppm; ¹³C NMR(125 MHz, CD₃OD) δ 156.9, 153.8, 149.9, 140.7, 118.1, 89.4, 82.6 (d,J=7.7 Hz), 76.6, 66.9 (t, J=4.9 Hz), 64.2 (q, J=5.2, 4.5 Hz) (2C), 63.6,50.5, 24.9 (3C), 17.5, 15.9, 15.0 (d, J=3.5 Hz), 14.9 (d, J=3.8 Hz),−5.2, −5.6 ppm; HRMS calcd for: C₂₂H₄₀ClN₅O₇PSi [M+H]⁺: 580.2118; found580.2121 (0.54 ppm).

Example 2.47—Intermediate Compound

2,5-dioxopyrrolidin-1-yl 4-(4-(prop-2-yn-1-yloxy)benzoyl)benzoate

To a solution of the corresponding acid^(18,19) (480 mg, 1.71 mmol) inDCM (10 mL, 0.17 M) was added N-hydroxysuccinimide (221 mg, 1.92 mmol)and EDC-HCl (368 mg, 1.92 mmol) at room temperature and the mixture wasstirred for 16 hours, before addition of H₂O and Et₂O. The aqueous layerwas extracted with Et₂O (2×) and the combined organic fractions werewashed with brine, dried (MgSO₄), filtered and concentrated in vacuo.Crude product was purified by flash chromatography on silica gel(Hexanes/Et₂O, 50:50-Et₂O 100%) to give the product (500 mg, 77% yield)as a pale yellow solid. R_(f)=0.25 (Et₂O 100%); Formula: C₂₁H₁₅NO₆; MW:377.35 g/mol; IR (neat) v_(max) 3300, 1725, 1325, 1180 cm⁻¹; ¹H NMR (500MHz, CDCl₃) δ 8.27 (d, J=8.1 Hz, 2H), 7.87 (d, J=8.2 Hz, 2H), 7.84 (d,J=8.8 Hz, 2H), 7.09 (d, J=8.7 Hz, 2H), 4.81 (d, J=2.3 Hz, 2H), 2.95 (s,4H), 2.59 (t, J=2.3 Hz, 1H) ppm; ¹³C NMR (125 MHz, CDCl₃) δ 194.2, 169.0(2C), 161.6, 161.3, 143.7, 132.5 (2C), 130.5 (2C), 130.0, 129.7 (2C),127.7, 114.7 (2C), 77.6, 76.4, 55.9, 25.7 (2C) ppm; HRMS calcd for:C₂₁H₁₅NO₆Na [M+Na]⁺: 400.0791; found 400.0797 (1.29 ppm).

Example 2.48—Cardioprotective Compound-LCB2191

((2R,3R,4S,5R)-2-(6-amino-2-chloro-9H-purin-9-yl)-5-(((diethoxyphosphoryl)oxy)methyl)-4-hydroxy-3-methyltetrahydrofuran-3-yl)methyl4-(4-(prop-2-yn-1-yloxy)benzoyl)benzoate (LCB2191)

To a solution of nucleoside (26.9 mg, 0.0464 mmol) in THF (1 mL) wasadded t-BuMgCl (64.9 μL, 64.9 μmol) dropwise at room temperature and themixture was stirred for 45 minutes. The photoaffinity probe (22.7 mg,60.3 μmol) was added slowly and the reaction was stirred for 16 hours.The reaction was quenched by addition of MeOH and concentrated in vacuo.The TBS-protected intermediate was obtained as a white powder and thecrude mixture was then used as such for the next reaction. To the crudemixture (12 mg, 14.2 μmol) in THF (142 μL, 0.1 M) at 0° C. was slowlyadded TBAF (21.4 μL, 21.4 μmol), then the mixture was warmed to roomtemperature and stirred for 16 hours. The reaction was quenched with asaturated solution of NaHCO₃ and the aqueous layer was extracted withEtOAc (3×). The combined organic fractions were dried (MgSO₄), filteredand concentrated in vacuo. Crude product was purified by reverse phase(C18) flash chromatography (H₂O/MeCN, 60:40) to give the final productas a white solid (5.3 mg, 47% yield over 2 steps). R_(f)=0.3 (DCM/MeOH,90:10); Formula: C₃₃H₃₅ClN₅O₁₀P; MW: 728.09 g/mol; IR (neat) v_(max)3285, 2979, 2904, 2571, 2345, 1719, 1605, 1454, 1266 cm⁻¹; ¹H NMR (500MHz, CD₃OD) δ 8.31 (s, 1H), 8.22 (d, J=8.0 Hz, 2H), 7.85 (t, J=8.9 Hz,4H), 7.16 (d, J=8.7 Hz, 2H), 6.52 (s, 1H), 4.91 (d, J=2.3 Hz, 2H), 4.71(d, J=11.4 Hz, 1H), 4.54 (d, J=11.4 Hz, 1H), 4.50-4.48 (m, 2H), 4.42 (d,J=8.1 Hz, 1H), 4.30 (dd, J=8.0, 3.9 Hz, 1H), 4.23-4.13 (m, 4H), 3.05 (t,J=2.3 Hz, 1H), 1.38-1.31 (m, 6H), 0.97 (s, 3H) ppm (Labile protons werenot observed due to exchange with deuterated solvent); HRMS calcd for:C₃₃H₃₆ClN₅O₁₀P [M+H]⁺: 728.1883; found 728.1882 (−0.072 ppm).

Example 2.49—Intermediate Compound

2,5-dioxopyrrolidin-1-yl4-(1-(prop-2-yn-1-yl)-1H-indole-5-carbonyl)benzoate

To a solution of the corresponding acid²⁰ (506 mg, 1.67 mmol) in DCM (10mL, 0.17 M) was added N-hydroxysuccinimide (230 mg, 2.00 mmol) andEDC-HCl (358 mg, 1.87 mmol) at rt and the mixture was stirred for 16hours, before addition of H₂O. The aqueous layer was extracted with DCM(3×) and the combined organic fractions were washed with brine, dried(MgSO₄), filtered and concentrated in vacuo. Crude product was purifiedby flash chromatography on silica gel (Hexanes/EtOAc, 50:50) to give theproduct (257 mg, 39% yield) as a pale yellow foam. R_(f)=0.45(Hexanes/EtOAc, 30:70); Formula: C₂₃H₁₆N₂O₅; MW: 400.39 g/mol; IR (neat)v_(max) 3280, 1771, 1739, 1648, 1599, 1202 cm⁻¹; ¹H NMR (500 MHz, CDCl₃)δ 8.28 (d, J=8.6 Hz, 2H), 8.10 (dd, J=1.7, 0.6 Hz, 1H), 7.91 (d, J=8.6Hz, 2H), 7.85 (dd, J=8.7, 1.7 Hz, 1H), 7.52 (dt, J=8.7, 0.8 Hz, 1H),7.34 (d, J=3.2 Hz, 1H), 6.67 (dd, J=3.3, 0.8 Hz, 1H), 4.96 (d, J=2.6 Hz,2H), 2.96 (s, 4H), 2.48 (t, J=2.6 Hz, 1H) ppm; ¹³C NMR (125 MHz, CDCl₃)δ 195.8, 169.0 (2C), 161.4, 144.7, 138.4, 130.4 (2C), 129.8 (2C), 129.2,128.9, 128.3, 127.3, 125.8, 124.0, 109.6, 104.1, 76.9, 74.2, 36.2, 25.7(2C) ppm; HRMS calcd for: C₂₃H₁₆N₂NaO₅ [M+Na]⁺: 423.0951; found 423.0938(−3.26 ppm).

Example 2.50—Cardioprotective Compound-LCB2194

((2R,3R,4S,5R)-2-(6-amino-2-chloro-9H-purin-9-yl)-5-(((diethoxyphosphoryl)oxy)methyl)-4-hydroxy-3-methyltetrahydrofuran-3-yl)methyl4-(1-(prop-2-yn-1-yl)-1H-indole-5-carbonyl)benzoate (LCB2194)

To a solution of the TBS protected nucleoside (41.0 mg, 70.7 μmol) inTHF (1 mL) was added t-BuMgCl (98.9 μL, 98.9 μmol) dropwise at roomtemperature and the mixture was stirred for 45 minutes. Thephotoaffinity probe (31.1 mg, 77.7 μmol) was added slowly and thereaction stirred for 16 hours. The reaction was quenched by addition ofMeOH and concentrated in vacuo. The intermediate was obtained as a whitepowder and the crude mixture was then used as such for the nextreaction. To the crude mixture (40 mg, 46.2 μmol) in THF (460 μL, 0.1 M)at 0° C. was slowly added TBAF (69.3 μL, 69.3 μmol), then the mixturewas warmed to room temperature and stirred for 16 hours. The reactionwas quenched with a saturated solution of NaHCO₃ and the aqueous layerwas extracted with EtOAc (3×). The combined organic fractions were dried(MgSO₄), filtered and concentrated in vacuo. Crude product was purifiedby reverse phase (C18) flash chromatography (H₂O/MeCN, 60:40) to givethe final product as a white solid (20 mg, 38% yield over 2 steps).R_(f)=0.3 (DCM/MeOH, 90:10); [α]²⁵ _(D) −27 (c 0.35, MeOH); Formula:C₃₅H₃₆ClN₆O₉P; MW: 751.13 g/mol; IR (neat) v_(max) 3269, 2968, 2909,2362, 2329, 1722, 1612, 1316, 1259 cm⁻¹; ¹H NMR (500 MHz, CD₃OD) δ 8.31(s, 1H), 8.24 (d, J=7.8 Hz, 2H), 8.09 (d, J=1.8 Hz, 1H), 7.86 (d, J=7.9Hz, 2H), 7.78 (dd, J=8.6, 1.6 Hz, 1H), 7.63 (dd, J=8.7, 0.9 Hz, 1H),7.45 (d, J=3.3 Hz, 1H), 6.66 (dd, J=3.3, 0.8 Hz, 1H), 6.52 (s, 1H), 5.09(d, J=2.5 Hz, 2H), 4.69 (d, J=11.4 Hz, 1H), 4.54 (d, J=11.5 Hz, 1H),4.52-4.47 (m, 2H), 4.42 (d, J=8.3 Hz, 1H), 4.31 (dd, J=8.0, 3.9 Hz, 1H),4.22-4.13 (m, 4H), 2.90 (t, J=2.5 Hz, 1H), 1.34 (tdd, J=7.0, 5.2, 1.0Hz, 6H), 0.96 (s, 3H) ppm (Labile protons were not observed due toexchange with deuterated solvent); ¹³C NMR (125 MHz, CD₃OD) δ 196.9,165.7, 156.7, 154.1, 150.2, 142.9, 139.6, 138.6, 132.2, 129.5, 129.4(2C), 129.2 (2C), 128.5, 128.3, 125.2, 123.3, 117.6, 109.5, 103.3, 88.6,82.1 (d, J=8.0 Hz), 77.4, 74.9, 73.6, 66.8, 66.3 (d, J=4.1 Hz), 64.4 (d,J=2.9 Hz), 64.3 (d, J=2.7 Hz), 49.1, 35.2, 16.1, 15.1 (d, J=2.9 Hz),15.0 (d, J=3.0 Hz) ppm; HRMS calcd for: C₃₅H₃₇ClN₆O₉P [M+H]⁺: 751.2043;found 751.2043 (0.078 ppm).

REFERENCES

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1. A compound of formula:

or a pharmaceutically acceptable salt thereof, wherein: A and B areC₁-C₆ alkyl, mono- to per-halo C₁-C₆ alkyl, —(CH₂)_(n)M, —C≡N, or

 with the proviso that: A is different from B, when one of A and B ismethyl, the other is not —CF₃, and when one of A and B is C₂-C₆ alkyl,the other is not C₂-C₆ fluoroalkyl; n is 1 to 3; R₁ is

R₂ is the same or different and is C₁-C₆ alkyl; M is —OR₃, —SR₃, aryl,—C(O)OR₃, or —OC(O)R₄; R₃ is —H, C₁-C₆ alkyl, aryl, aryl-C₁-C₆ alkyl,C₁-C₆ alkylaryl, wherein each of the alkyl and aryl groups is optionallysubstituted with one or more groups selected from halo, mono- toper-halo C₁-C₆ alkyl, —CN, —C(O)OH, —C(O)OR₄, —N₃, —C₁-C₆ alkyl-C(O)OR₄,—CF₃, —C₁-C₆ alkyl-N₃, and —SiF₅; R₄ is C₁-C₆ alkyl, aryl, heteroaryl,C₁-C₆ alkylaryl, aryl-C₁-C₆ alkyl, wherein each of the alkyl, aryl andheteroaryl groups is optionally substituted with one or more groupsselected from halo, —CN, alkynyl, alkynyloxy, —C(O)OH, —N₃, —CF₃, —C₁-C₆alkyl-N₃, —SiF₅, —NH₂, and —NHR₃; C and D are independently —H, halo,azido, —OR₃, —CN, or —CF₃; X is O or S; and Base is:

R₅ is —H, —C(O)—C₁-C₄ alkyl, aryl, alkylaryl, or arylalkyl, wherein eachof the alkyl and aryl group is optionally substituted with one or moregroups selected from halo, —R₄, —CF₃, and —N₃.
 2. (canceled) 3.(canceled)
 4. The compound of claim 1 being of the formulae:


5. (canceled)
 6. The compound of claim 1, wherein one of A or B is C₁-C₆alkyl while the other is —(CH₂)_(n)M, —C≡N, or


7. (canceled)
 8. (canceled)
 9. The compound of claim 1, wherein n is 1.10. The compound of claim 1, wherein M is —OR₃ or —OC(O)R₄. 11.(canceled)
 12. (canceled)
 13. The compound of claim 10, wherein R₃ is—H, C₁-C₆ alkyl, or aryl-C₁-C₆ alkyl, wherein the aryl of the aryl-C₁-C₆alkyl is optionally substituted with one or more: halo, mono- toper-halo C₁-C₆ alkyl, —N₃, and/or —C₁-C₆ alkyl-N₃.
 14. (canceled) 15.(canceled)
 16. (canceled)
 17. (canceled)
 18. (canceled)
 19. The compoundof claim 13, wherein R₃ is —H, methyl, isopropyl, benzyl,


20. (canceled)
 21. (canceled)
 22. The compound of claim 10, wherein, in—OC(O)R₄ in M, R₄ is aryl or heteroaryl, the aryl and heteroaryl beingoptionally substituted with one or more groups selected from halo, —CN,alkynyl, alkynyloxy, —C(O)OH, —N₃, —CF₃, —C₁-C₆ alkyl-N₃, —SiF₅, —NH₂,and —NHR₃.
 23. (canceled)
 24. (canceled)
 25. (canceled)
 26. (canceled)27. (canceled)
 28. (canceled)
 29. (canceled)
 30. The compound of claim1, wherein, in —OC(O)R₄ in M, R₄ is


31. (canceled)
 32. (canceled)
 33. The compound of claim 1, wherein oneof C or D is —H and the other is halo or —OR₃.
 34. (canceled) 35.(canceled)
 36. (canceled)
 37. (canceled)
 38. The compound of claim 1,wherein X is O.
 39. The compound of claim 1, wherein R₅ represents —H,—C(O)—C₁-C₄ alkyl, arylalkyl, or aryl, wherein the aryl group isoptionally substituted with one or more groups selected from halo, —R₄,—CF₃, and —N₃.
 40. (canceled)
 41. (canceled)
 42. (canceled) 43.(canceled)
 44. (canceled)
 45. (canceled)
 46. (canceled)
 47. (canceled)48. (canceled)
 49. (canceled)
 50. The compound of claim 1, wherein baseis:

51-78. (canceled)
 79. The compound of claim 1 being:

or a pharmaceutically acceptable salt thereof.
 80. (canceled) 81.(canceled)
 82. The compound of claim 79 being

or a pharmaceutically acceptable salt thereof.
 83. (canceled)
 84. Amethod of providing cardioprotection in a subject in need thereof, themethod comprising administering the compound of claim 1 or apharmaceutically acceptable salt thereof to the subject.
 85. A method ofpreserving, reducing deterioration of, and/or improving a cardiacfunction of a heart that has been subjected, is subjected, or will besubjected to a cardiac insult, the method comprising administering thecompound of claim 1 or a pharmaceutically acceptable salt thereof to asubject in need thereof.
 86. (canceled)
 87. A method of preventing,reducing, and/or reversing heart damage due to a cardiac insult, themethod comprising administering the compound of claim 1 or apharmaceutically acceptable salt thereof to a subject in need thereof.88. (canceled)
 89. A method preventing and/or treating of a cardiacdysfunction due, at least in part, to a cardiac insult, the methodcomprising administering the compound of claim 1 or a pharmaceuticallyacceptable salt thereof to a subject in need thereof.
 90. (canceled) 91.(canceled)
 92. A method preventing and/or reducing cardiotoxicityassociated with use of a cardiotoxic drug, and/or reversing thecardiotoxic effects thereof, the method comprising administering thecompound of claim 1 or a pharmaceutically acceptable salt thereof to asubject in need thereof.
 93. (canceled)
 94. (canceled)